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This is to certify that the
dissertation entitled

C(NTROL OF CDS EXPRESSICNQNI
MURINE B-LINEAGE CELLS

presented by

Timothy Raymond Weichert

has been accepted towards fulfillment
of the requirements for

Ph-D- degree in W

 

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This is to certify that the

dissertation entitled

CCNTROL OF CD5 EXPRESSIG‘ION
MURINE B—LINEAGE CELLS

presented by

Timothy Raymond Weichert

has been accepted towards fulfillment
of the requirements for

Ph-n- degreein WY

\

.1111! -_
Majorpro .

Date April 7, 1994

MS U is an Affirmative Action/Equal Opportunity Institution

 

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MSU to An Affirmative Adlai/Emu Opportunity Inetltulon
Wm:

CONTROL OF CD5 EXPRESSION
ON MURINE B-LINEAGE CELLS

BY

Timothy Raymond Weichert

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1994

ABSTRACT

CONTROL OF CD5 EXPRESSION
ON MURINE B-LINEAGE CELLS

BY
Timothy Raymond Weichert

The CD5 surface antigen is expressed on all T-cells and
on a subset of B-cells found mainly in serousal cavities of
adult mice. It has previously been shown that adult murine
bone marrow does not contain progenitors for CD5+ B-cells,
prompting some investigators to classify CD5+ B-lymphocytes as
a unique lineage of B-cells. However, we have found that the
v-Ha-rag and v-sgg oncogenes, but not v—myg, can induce the
expression of CD5 on two bone marrow-derived pre—B-cell lines
not previously expressing this marker, and that this up-
regulation correlates with increased steady-state levels of
CD5 mRNA. This suggests that the CD5 phenotype is available
to CD5“ B-lineage cells and may alter the concept that CD5-
expressing B-cells represent a unique, self-renewing subset of
B-lymphocytes.

The lymphoid-specific expression of CD5, along with its
up-regulation by transforming oncogenes, suggests that
interesting mechanisms control the expression of CD5. To
identify these mechanisms, we have cloned and sequenced over
1100 base pairs of the murine CD5 5' -flanking region, isolated
from an NIH 3T3 fibroblast genomic library. The CD5 5' -
flanking region is capable of a lymphoid-specific pattern of

promoter activity that reflects in vivo CD5 expression. The

CD5 promoter lacks a consensus TATA box and initiates
transcription from multiple sites around an initiator (INR)—
like element. These characteristics may' place the CD5
promoter in the same class as the mb-l and B29 lymphoid-

specific TATA-less promoters.

This Dissertation is Dedicated to My Beloved Wife
Michelle Anderson
and
To My Parents

Donald and Lois weichert

iv

TABLE OF CONTENTS

List of Figures
Introduction
Chapter 1 Literature Review

1. The CD5 Surface Antigen
1.1 Molecular Aspects of CD5 .
1.2 Function of the CD5 Molecule .
2. Characteristics of B- lymphocyte Subsets
Expressing CD5
2.1 CD5 as a Marker for a Distinct Lineage of
B- lymphocytes .
2.2 Surface Markers of the CD5*ZB- cell .
2.3 CDS- -Expressing B- cells Have Monocytoid
Features . . . . . . . . .
2. 4 CD5 B- cells in Disease
2.4.1 Autoimmunity, Autoantibodies and
Natural Antibodies
2. 4. 2 CD5 B- cells in Chronic Lymphocytic
Leukemia and Neoplastic Transformation
3. The Role of Oncogenes in B—cell Transformation
3.1 The abl Oncogene .
3.2 The gr; Family of Tyrosine .Kinases
3.3 The my; Oncogene
3.4 The ras Oncogene .
4. Possible Origins of CD5‘IB- lymphocytes
4.1 The Modulation of CD5 Expression
4.2 CD5’ B-cells Are Made, Not Born
4.3 A Thesis Proposal . . . .
Literature Cited

Chapter 2 Cloning and Tissue- —Specific Regulation

of the Murine CD5 Promoter

Abstract

Introduction
Materials and Methods
Results

Discussion
References

vii

13
15
16
17
18
21
24
24
26
27
29

37

38
39
41
47
72
75

Chapter 3 Regulation of Murine CD5 Expression
by Transforming Oncogenes . . . . . . . . . . . 77

Abstract . . . . . . . . . . . . . . . . . . . . . . 78
Introduction . . . . . . . . . . . . . . . . . . . . 79
Results . . . . . . . . . . . . . . . . . . . . . . 84
Discussion . . . . . . . . . . . . . . . . . . . . 103
Materials and Methods . . . . . . . . . . . . . . 108
References . . . . . . . . . . . . . . . . . . . . 114

Summary . . . . . . . . . . . . . . . . . . . . . . . . 117
References . . . . . . . . . . . . . . . . . . . . 120

vi

Chapter

1.

LIST OF FIGURES

2

Cloning Strategy and Partial Restriction

Map of the CD5 5’-Flanking Region

Sequence of the CD5 5’-F1anking Region

The 5’ -Termini of CD5 mRNA Map to the

CD5 5'- -Flanking Region . . . . . .
Restriction Map of the CD5. 5’- -Flanking
Region Deletion Constructs

Tissue- Specific Expression of the CD5
Promoter

Maximal CD5 Promoter Activity is Orientation-
Dependent

Activity of CD5 Promoter Deletion Constructs
in EL4 Cells

Activity of CD5 Promoter Deletion Constructs
in M12 Cells

Activity of CD5 Promoter Deletion Constructs
in NIH 3T3 Fibroblasts

3

FACS analysis of CD5 surface expression on

R3 and RM2 cell lines

FACS analysis of CD5 surface expression on
high density bone marrow (HDBM) cultures
infected with v- H- ras . .

FACS analysis of CD5 surface expression on
Clone 3 cells infected with v- -H- ras

Northern analysis of CD5 expression in Clone 3
cells infected with v- -H- ras .

FACS analysis of CD5 surface expression on PD36
cells transfected with pSV(X)- -H- ras

FACS analysis of CD5 surface expression on M12
cells transfected with pSV(X)- -H- ras . .
Northern analyis of PD36 cells infected with
v- -H- ras, v- myc, A- MuLV, v- src and MoMuLV
Northern analysis of PD36 cells stably
transfected with pSV(X)- -H- ras

Activity of the CD5 promoter in PD36ras

cells . . . . . . . . . . .

vii

44
48

51

55

58

60

64

66

70

86

87

89

91

93

94

97

99

102

INTRODUCTION

Our initial involvement with the CD5 surface antigen
occurred during the ’standard' phenotypic analysis of four
pre-B-cell populations independently derived by 'v—Ha-rag
infection of high density murine bone marrow cultures (R1-4;
Schwartz et al., 1986). Each.population expressed CD5. A few
years earlier, an elegant experiment by Hayakawa, et al.
(1985) had established CD5+ B-cells as a unique lineage of B-
cells. They found that bone marrow could not replenish the
CDS“ B-cell population in lethally—irradiated mice, suggesting
that the progenitors of CD5+ B-cells are not found in bone
marrow. Yet, we possessed four transformed bone marrow-
derived pre-B-cell populations that expressed CD5. A
literature review' demonstrated that other labs had. also
reported frequent establishment of<3I?'pre-B-cell lines from
bone'marrowwutilizing'transforming'oncogenes (Davidson.et.al.,
1984; Holmes et al., 1986). A.closer study of the literature,
along with a survey of transformed cell lines in our lab,
yielded a surprising result. E_ve_r_y pre-B-cell population
established with v-Ha-rgg expressed CD5. To reconcile the
transformation data with the lineage model, one would have to
conclude that minor, contaminating populations of CD5” B-

lineage cells in the bone marrow are preferentially

2

susceptible to transformation by the v—Ha—;§§_oncogene during
each. independent infection. This seemed. 'unlikely.
Alternatively, the targets of the virus may include CDS‘ B-
cells. If this was occuring, it was formally possible that
the v-Ha-ggg oncogene was inducing the expression of CD5 on
jpreviously'CDS‘ cells. This has turned out to be the case»

Chapter 1 of this thesis will detail the more popular
theories of CD5+ B-cell lineage descent, as well as, the
current status of the field” Chapter 2 details the cloning of
the murine CD5 5’—flanking region and demonstrates that it is
capable of a pattern of tissue-specific expression reflective
of in vivo CD5 expression. Chapter 3 will demonstrate that
the v-Ha-r§§_oncogene is capable of inducing the expression.of
CD5 on pre—B-cell lines. In the summary section, the status
of the CD5 field will be viewed from the perspective of our

findings.

CHAPTER 1
LITERATURE REVIEW

The assignment of unique lineage status to CD5-expressing
B-lymphocytes has proven to be one of the most divisive
controversies in immunology in the last ten years. Several
unifying theories addressing the origin of CD5+ B-cells have
been advanced, each.depending upon either the co-expression of
ill-defined surface antigens or strict anatomical
compartmentalization of lymphoid subsets to make its case.
This chapter will summarize the characteristics of CD5
expressing B—cells, with a review of the more popular theories
of their lineage descent, and will ultimately present data
suggesting that the CD5 phenotype is available to a range of

B—lymphocytes and is not a lineage-restricted phenomenon.

1. The CD5 Surface Antigen

1.1 Molecular Aspects of CD5

The CD5 (Ly-1, Leu-l, Lyt-l) surface antigen is a 67 kDa
MW monomeric, membrane-associated glycoprotein, expressed on
all T-lymphocytes and a subset of B—lymphocytes (Huang et al,
1987; Jones et al, 1986). CD5 was originally defined by the
monoclonal antibody (mAb) OKT-l, which was produced from a
hybridoma generated by the fusion of myeloma cells with spleen
cells from mice immunized with human T-cells (Reinherz et al,
1979). Eventually, monoclonal antibodies were used to purify
the CD5 protein allowing the N-terminal amino acids to be

sequenced. Degenerate oligonucleotides to this sequence were

4
synthesized and used to probe a human T-cell cDNA library
which yielded the human cDNA clone (Jones et al., 1986). The
human cDNA.was used to probe a murine T-cell cDNA library and
the murine CD5 cDNA was isolated (Huang et al., 1987).

The murine cDNA is 2.2 kilobases (Kb) in length and has
an open reading frame of 1482 bases coding for a protein of
494 amino acids (Huang et al., 1987). The full-length cDNA
includes a 3’ untranslated region of 457 base pairs (bp) and
a 5’ region upstream of the terminal methionine codon of only
6 bp» The first 23 amino acids have leader sequence homology,
while residues 378 to 391 have the strong hydrophobic profile
of a transmembrane sequence. Altogether, the CD5 protein
consists of an N-terminal region of 347 amino acids, a 30
residue transmembrane region and a 94 residue carboxy-terminal
domain. Initially, the protein was predicted to have folding
homology'to the hinge region.of immunoglobulin (Ig) molecules,
but recent work has shown greater identity to the cysteine-
rich scavenger receptor family of polyanion-binding membrane-

associated proteins (Freeman et al., 1990).

1;; Function of th§:CD5 Molecule

In the first ten years following it's discovery, the
number of functions attributed to the CD5 molecule was
directly proportional to the number of investigators
addressing the question. Alternate hypotheses have included
CD5 as an activation antigen, a tumour neo-antigen and a

marker defining a sub-lineage of B-lymphocytes (for review,

5
see Kipps, 1989). Until recently, very few investigators have
offered rigorous analyses addressing the involvement of the
CD5 molecule itself in the physiology of lymphocytes.

Van de velde et al. (1991), using biotin-labelled CD5
protein, identified a B-cell-specific surface antigen, CD72
(Lyb-2), as a ligand for CD5. This may implicate CD5 in T—
cell/B-cell interactions as well as B-cell/B-cell
interactions.

The carboxy-terminal cytoplasmic tail of the CD5 coding
region contains many predicted sites for protein kinase C
(PKC)-mediated serine/threonine phosphorylation as well as
sequences homologous to the tyrosine autophosphorylation sites
of the m, 115. and f3}; oncoproteins (Huang et al., 1987;
Chatila and Geha, 1988). Chatila and Geha (1988) have shown
that activation of PKC ‘with. phorbol esters does indeed
hyperphosphorylate CD5, implicating it in the transduction of
extra-cellular signals (also Berridge, 1984).

The use of anti-CD5 mAb's as substitute ligands has shown
that CD5 can transduce a proliferation signal in T-cells,
possibly by up-regulating IL-2 and IL-2 receptors (Ledbetter
et al., 1985; Ceuppens and Baroja, 1986; Spertini et al.,
1991) , and increase the mobilization of extra-cellular calcium
in T-cells (June et al., 1987). Since these activities have
been observed only in TCR*/CD3*/CD5” T-cells, and since
activation of the TCR/CD3 complex induces T-cell activation
and increased calcium flux, it was postulated that CD5 signal

transduction may be integral in the TCR/CD3-mediated

6
proliferation response. 'This idea is supported.by the work.of
Burgess et al. (1992), which showed that anti-CD5 mAbs could
co-precipitate a CDS/TCR/CD3 complex that includes the protein
tyrosine kinases p5610k and p59fY", which may be responsible for
phosphorylating CD5 (also Osman et al. , 1992) . While no
parallels have been found in B-cell-associated CD5, the recent
placement of CD5 in the scavenger receptor family of receptors
may implicate CD5 in the binding of modified proteins (i.e.

acetylated lipoproteins) (Freeman et al., 1990).

2. Characteristics of B-lvgphoczte Subsgts Exprggsing CD5
2.1 CD5 as a Marker for a Distinct Lineage of B-lzgphocxteg

As stated previously, CD5 is a pan-T-cell marker and is

 

expressed on a minority of B-cells. In normal animals, the
distribution of CD5+ B-cells appears to be compartmentalized.
About thirty percent of all lymphocytes in the murine and
human.peritoneal, pleural and pericardial cavities are CD5‘II-
lymphocytes, with small populations found in murine spleen,
human lymph node and human tonsil (Kipps, 1989; Kroemer et
al., 1993). CD5*IB-cells are not found in adult bone marrow
or the peripheral circulation except in certain autoimmune
diseases and chronic lymphocytic leukemia (CLL). This
intriguing distribution in normal animals and apparent
breakdown of compartmentalization in disease states that
involve immune dysregulation, have led to the hypothesis that
CD5+ B-lymphocytes may be a lineage distinct from peripherally

located CDS' B-cells.

7

The above hypothesis was apparently supported by a now—
classic experiment by Hayakawa et al. (1985) . In their
report, the authors used adult murine bone marrow cells to
reconstitute the immune system of lethally-irradiated mice and
subsequently could not detect CD5+ B-cells in the recipients.
However, if peritoneal cells were used in the reconstitution,
donor CD5+ B-cells could eventually be found to repopulate the
peritoneal cavity. Herzenberg et al. (1986) , have interpreted
this to mean that CD5+ B-cells can only be replenished from
CD5’IgM” precursors and not from CDS’IgM' pro/pre-B-cells found
in adult murine bone marrow. A related experiment utilized a
strain of mice whose B-cells expressed either surface IgHA or
IgHB heavy chain alleles. The authors then injected the mice
with anti-IgHB mAb to deplete this set of mature B-cells.
Even after a year, FACS (fluorescence—activated cell sorting)
analysis could not detect IgHBCDS+ B-cells in any compartment.
This suggests that while bone marrow could replenish IgHB B-
cells from sIgM‘ precursors, it could not renew the CD5+ B-
cells because their precursors reside outside the marrow as
IgM"CD5+ cells (Lalor et al. , 1989) . Further permutations of
these adoptive transfer experiments have found that fetal
liver contains precursors for both CD5” and CD5‘ cells but
fetal omentum can only replenish CD5+ cells (for review, see
Kroemer et al. , 1993) . In contrast to the above, Iacomini and
Imanishi-Kari (1992) have repeated the bone marrow adoptive
transfer experiments of Hayakawa et al. (1985) and found that

CD5+ B-cells could be repopulated in lethally—irradiated mice

8
from donor bone marrow cells. This discrepant finding of CD5+
precursors in adult bone marrow has not been satisfactorily

explained at this time.

2.2 Surface Markers of the CD5” B-cell

Upon examination of the populations that reconstitute
CD5‘ B-cells in adoptive transfer experiments, a pattern of
commonly expressed surface antigens has emerged. The
phenotype of CD5+ B-lymphocytes is generally accepted to be
c135+IthighlgD1°"Mac-1*CD451°"1~"c.r~3-R'IL-5R+ which is opposite of
'normal' B—cells (Stall et al., 1992; waldschmidt et en”,
1992; Kroemer et al., 1993). During these adoptive transfer
experiments, some investigators noted that donor cells from
compartments that reconstitute CD5+ B-cells also reconstitute
B-cells with the above surface phenotype that were CD5“ (Stall
et al., 1992). This population, termed the CD5 ’sister ’
population, only differs in that CD5 is poorly expressed or
absent. This prompted a group of senior CD5 B-cell
investigators to reclassify all B-cells as either B1-
1ymphocytes (CD5+ B-cells [B-1a] and the CD5“ 'sister’
population. [B-lb]) or IB2-lymphocytes (all other' B-cells)
(personal account of the conference "CD5 B-cells in
Development and.Disease", PalulBeach.Gardens, Florida, June 3-
6, 1991). This nomenclature will not be heavily relied upon

in this paper.

9
2.3 CD5-Expressing B-cells Have Monocytoid Features

CD5+ B—cells tend to be adherent and occupy serousal
cavities, much like macrophages (Herzenberg and Stall, 1989;
Hayakawa et al., 1986). These B-cells express the classical
macrophage markers CD11/MAC1 (C3R), CDllb, CD14, and CD15 as
well as the B-specific antigens CD19, CD20, CD21 and CD72
(Kipps, 1989; Herzenberg et al., 1987).

Certain CDS” myeloid lines have rearranged Ig genes
suggesting that they either differentiated from pre-B-cell
precursors or have superimposed developmental programs (Kemp
et al., 1980). Likewise, all pre-B-cell lines that can
differentiate into macrophage lines have been found to be CD5+
in cases where this marker has been examined (Bretz et al.,
1992; Holmes et al., 1986; Davidson et al., 1988). The
establishment of CD5+ pre-B-cell lines with myeloid
characteristics was interpreted by some to mean that monocytic
features are a distinct trait of the unique CD5 lineage

(Herzenberg and Stall, 1989).

MES B-cells in Disease

2.4.1 Autoimmunity, Autoantibodies and Natural Antibodies
Flow cytometric studies have detected increased levels of

CD5“ B-cells in the circulation of patients with rheumatoid

arthritis and primary Sjogren’s syndrome (Hardy et a1. , 1987;

Toniguchi et al. , 1987; Lydyard et al. , 1987; Dauphine et al. ,

1988) . CD5+ B-cells are also found in increased numbers in

systemic lupus erythematosis (SLE) ~prone NZBxNZW mice (Shirai

10

et al. , 1991) . These autoimmune diseases are characterized by
pathological tissue destruction thought to be mediated by
autoreactive antibodies. The CD5+ B-cell was implicated as a
source of this autoantibody when sorted populations of this
cell type, taken from rheumatoid arthritis patients, were
found to secrete rheumatoid factor (an anti-IgG IgM) (Hardy et
al., 1987) . Subsequently, CDS“ B-cells were found to secrete
IgM-class antibodies to single-stranded DNA, bromelain-treated
erythrocytes, thymocytes, thyroglobulin, with some having
cross-reactivity to bacterial coat proteins and herpes simplex
glycoproteins (Calvanico, 1993; Kipps, 1989; Mayer et al.,
1990; Shirai et al., 1991).

The basis for the CD5+ B-cell's Ig repertoire being

limited to production of autoantibody lies in it’s restricted

use of Ig variable region genes (Mercolino et al. , 1986;
Pennel et al., 1988). In support of this, Reininger et al.
(1987) , described three clonally unrelated, CD5‘, anti-

erythrocyte antibody-producing hybridomas, generated from NZB
mice, that utilized identical variable heavy chain Ig genes
and nearly identical variable kappa light chain Ig genes.
This work was supported by that of Forster et al. (1988),
which describes the generation of seventeen CD5+ hybridomas
with similar specificities. Surprisingly, the CD5+ CH series
of lymphomas (isolated from aging mice hyper-immunized with
sheep erythrocytes) also rearranged the identical IgH variable
regions as the autoantibody-producing hybridomas previously

mentioned (Kofler, 1988; Winter et al., 1985). These studies

11

suggest that in vitro methods can be used to study CD5* B-cell
variable chain usage. Moreover, all the sequence analyses
available indicate that the Ig genes that show restricted use
in hybridomas have not significantly diverged from the
germline (for review, see Kipps, 1989). 13y all accounts,
selection of variable region genes in CD5+ B-cells is probably
not a random event and has prompted some authors to include it
as another identifying characteristic of the CD5” B-cell
lineage (Kantor and Herzenberg, 1993).

CD5+ B-cell-produced autoantibodies generally have low
binding affinities and multiple antigenic specificities and
are not the monoreactive, highly specific, IgG class
antibodies thought to be needed to convey autoimmune tissue
destruction. Evidence against CD5+ B-cells producing the
autoantibodies actually responsible for autoimmune pathology
began to mount when.it became apparent that the development of
clinically significant autoimmune disease in NZBxNZW mice
correlated with an isotype shift to IgG anti-DNA antibodies
produced by CD5' cells (Shirai et al., 1991) . It is not clear
if this was due to expansion of anti-DNA specific CDS' B-cells
or class-switching of the IgM anti-DNA CD5+ cells with
subsequent CD5 down-regulation.

When it became apparent that CD5+ B-cells may be
responsible for the production of autoreactive low—affinity
IgM and not pathologic high-affinity IgG autoantibody,
questions arose as to the physiological significance of these

self—directed antibodies. With data demonstrating that CD5+

12
B-cells populate the fetal mouse peritoneal cavity early in
ontogeny, Kipps (1989) suggested that self-directed, low—
affinity antibody could "establiSh a primordial network of
idiotypic and anti-idiotypic interactions" to stimulate clonal
expansion of certain B-cell subsets in the sterile fetal
environment (also Jerne, 1984; 1985). In the adult, CD5*IB-
cells could produce autoantibody to form immune complexes or
regulate post-thymic maturation of the T-cell antigen
repertoire (Martinez et al., 1986; Kipps, 1989). The
persistence of this autoantibody production from the neonatal
animal into the adult may also form the basis for ’natural'
immunity (humoural). These ’natural’ autoantibodies may take
part in removal of damaged. tissue, block cross-reactive
epitopes shared. between. host and invading' pathogens, or
provide a pool of non—specific antibodies that rapidly react
to microbial invasion while the process of class-switching and
antibody affinity—maturation is occuring (Logtenberg, 1990).
While the concept of ’natural’ autoantibody'production as the
specialized immune function of CD5‘IB-cells is an attractive
hypothesis, Kasaian.et al. (1992), have reported.that Epstein-
Barr virus (EBV) -transformed CDS' B-lymphocytes can be induced
to express 'natural' antibodies to ssDNA, insulin,
phosphorylcholine and tetanus toxoid” This suggests that CD5‘
B-Cells do not have a unique immune function and somewhat

detracts from their status as a separate B-lymphocyte lineage.

13

2.4.2 CD5 B-cells in. Chronic Lypphocytic Leukemia. and

 

Neoplastic Transformation

Chronic lymphocytic leukemia (CLL) is the most common
adult leukemia and.is characterized.by invasion.of blood, bone
marrow, lymph node and spleen by small B—cells (95% of cases)
or 'T-cells (5% of cases) (Deissorth et al., 1992). In B-CLL,
the neoplastic cells are clonal in nature and invariably
express CD5 on their surface (Freedman, 1990). These cells
also express surface IgM and other markers reflective of B-1
lymphocytes (1 . e. CD5+IthighIgD1°"Mac - 1*CD4 51°"FcE-R’IL- 512+ )
rather than B-2 cells (Freedman, 1990). Kipps (1988) found
that about 25% of CLL’s express autoantibody to determinants
that autoantibodies from murine CD5+ B-cells also identify.
Thus, B-CLL is thought to be the neoplastic counterpart of
naturally occuring ’Bla'-cells. Not coincidentally, there is
a large body of literature dealing with the expression of CD5
on transformed B-lineage cells.

The first report of CD5 expression on B-cells actually
involved spontaneously arising B-lymphomas from New Zealand
and.B—1O mice (Lanier, et a1, 1981). Soon after, Braun (1983)
reported a simple, long term splenocyte culture system that
yielded oligoclonal outgrowths of immortalized CDS‘IB-cells.
Davidson et al. (1984) described.a series of ten spontaneously'
occuring or deoney murine leukemia virus (MoMuLV)-induced
CD5+ B-lineage lymphomas that spanned the stages of chell
differentiation. Palacios et al. (1987) were able to devise

a culture system that allowed the establishment of CD5+ pro-B

l4
and.B-lymphocytes from.the bone marrow of four week old.Balb/c
mice.

Braun et al. (1986) subsequently found that the c-rnyc
proto-oncogene is over-expressed in the CDS“ lymphoblasts from
splenic cultures. This was followed.by anecdotal reports from
other investigators that also noted a preponderance of CD5*13
and pre-B—lymphocytes in culture systems that relied upon
transforming oncogenes for establishment of B-lineage lines
(Kipps, 1989; R. Schwartz, unpublished observations). Holmes
et al. (1986) authored. the seminal. paper‘ addressing" the
utilization of oncogene-expressing retroviruses to generate
CDS“ B-cell lines from murine bone marrow cultures. They
infected.bone marrow (also fetal liver cells) from 2 to 3 week
old NFS/N mice with Abelson murine leukemia virus (AeMuLV; v-
apl) or replication—defective retroviral vectors containing

the v-src, v-Ha-ras, K-ras or v-fes oncogenes. Cell lines

 

were established in liquid culture and then analyzed for CD5
expression via FACS analysis. It was found that v-apl and v-
gpg infected lines did not express or weakly expressed CD5,
while lines infected with v-Ha-pag vectors invariably
expressed this marker. The lines were predominantly pre-B-
cells regardless of tissue of origin. This result was
supported by Tidmarsh et al. (1989) who found zero out of
eleven cell lines generated by infection of bone marrow with
A-MuLV expressed CD5. Another group found that while 4 of 4
cell lines generated by v-Ha-pap infection of murine bone

marrow expressed CD5, 2 lines generated by v-Ha-ras/v-myc co-

15
infection did not (Schwartz et al., 1986, and unpublished

observations).

3. The Role of Oncogenes in B-cell Transformation

From the above, it appears that the CD6*IB—cell may be a
frequent target for' certain transforming' oncogenes (and,
presumably, their cellular homologs). Therefore, a brief
overview of the cellular targets, molecular biology, and
transforming characteristics of the aforementioned oncogenes
is in order.

Sawyers et al. (1991) define leukemia as "...the
uncontrolled proliferation or expansion of hematopoietic cells
that do not retain the capacity to differentiate normally to
mature blood cells." Implicit in this definition is that the
genetic events that cause leukemia, such as chromosomal
abberations and proto-oncogene activation, must dysregulate
both growth and differentiation. The support for this idea
(summarized below) has come from studies indicating that, in
general, oncogenes having hematopoietic neoplastic activity
confer either growth factor independence, arrest of
differentiation, or both. Moreover, the phenotype of the
arrested, transformed cell is thought to reflect the stage of
differentiation at which the normal cellular homolog of the

oncogene is active (for review, see Sawyer et al., 1991).

16

3.1 The abl Oncogene

The oncogene carried in the Abelson murine leukemia virus
(v-apl) has intrinsic tyrosine kinase activity, which is
undetectable in it’s cellular homolog c-apl (Witte et al.,
1980; Ponticelli et al., 1982). c-apl is closely related by
homology to the c-ppg family of tyrosine kinases, whose normal
cellular function is thought to entail signal transduction at
the cytoplasmic surface of the cell membrane. In t(9‘,22)
chromosomal translocations (Philadelphia chromosome) of human
chronic myelogenous leukemia (CML), mutant forms of c-apl are
able to display tyrosine kinase activity; Appearence of this
apl/bcr fusion product in CML is correlated with polyclonal
expansion of differentiated B-lymphoid and myeloid subsets
that may later evolve into a Iblast crisis of immature
progenitors (Sawyer et al., 1991). It is at the blast stage
that CML resembles murine Abelson disease in that the
aggressive lymphoid tumors can be characterized as lymphoid or
myeloid progenitors (Witte, 1983). To determine the lymphoid
target of the v-apl oncogene, Whitlock.et al. (1983) developed
an in vitro culture system that supports the growth of murine
bone marrow—derived early B-progenitors (perhaps even stem
cells), pre-B cells and B-cells. They then infected clonal
populations of B and pre-B—cells (derived by limiting
dilution) with vegpl and found that immature cell subsets
could still undergo differentiation to mature forms (analyzed
by subsequent Ig light chain gene rearrangement). This data,

combined with the fact that the Philadelphia chromosomal

l7
fusion is seen in all lymphoid subsets in CML, suggests that
the v-fl protein and the Lag/Q1 fusion protein target a very
early bone marrow progenitor, perhaps the stem cell. An
extension of this is that V-ibl dysregulates growth restraint

but not differentiation.

3.2 The src Family of Tyrosine Kinases

Other tyrosine kinases that can transform pre—B—cells
include v-fgg and v-ppg (Holmes et al., 1986). As members of
the v-ggg family of tyrosine kinases, they are intimately
associated with the inner leaf of the cell membrane via
myristic acid residues. They are endowed with a cytosolic
domain that is capable of down-regulating it's intrinsic
kinase activity by auto-phosphorylation.of a carboxy-terminal
tyrosine. The transforming activity associated.with v-gpg is
attributable to almissense mutation that replaces the carboxy-
terminal tyrosine with. another amino acid, resulting in
constitutive kinase activity. Probable implications of
constitutive activity are chronic activation of downstream
messengers such as phosphatidyl inositol 3-kinase and c-
;ap/GAP (for review see Cantley et al., 1991).

Recent work in the area of B—cell activation has
implicated the fig family of tyrosine kinases in signal
transduction via the B-cell receptors sIgM and sIgD.
Yaminishi et al. (1991) have shown that c-Lyp physically
associates with sIgM, while Burkhardt et a1. (1991) used

immunoprecipitation techniques to demonstrate interaction

18
between sIgD and c-plk, c-l , and c—fyp. Mizuguchi et al.
(1992) demonstrated that activating the B—cell by anti-IgM
cross-linking results in tyrosine phosphorylation, while Lin
and Justement (1992) suggested that this signal is transduced
through the sIg-asssociated proteins mb-l and 8-29.

Like v-apl transformations, the tumors derived from bone
marrow transformed with retroviruses expressing v-ppg are of
short latency (one to two weeks). In contrast, while v-apl
transformants may differentiate further into B-cells, cell
lines derived.by infection.with.v-f§§ or v-gpg are of a stable
pre-B or pre—B/myeloid phenotype (Pierce and Aaronson, 1983;

Pierce et al., 1984).

3.3 The myc Oncogene

The c-myg proto-oncogene product is a 49,000 MW protein
localized to the nucleus and is expressed in a myriad of cell
types (for review, see Bishop, 1991). It’s role in
hematopoietic neoplasia is best characterized by it’s
involvement in Burkitt’s lymphoma, a human B-cell lymphoma of
the peri-mandibular region and endemic to Africa.

c-pyg's involvement in Burkitt's lymphoma is thought to
begin after the clonal (or oligoclonal) outgrowth of Epstein-
Barr virus (EBV) -infected pro-B or pre-B-lymphocytes (Magrath,
1990). It is at this stage, where there is high
recombinational activity at the Ig heavy chain (pro-B) or
light chain (pre—B) loci, that rearrangement of the c-myp_gene

to an Ig promoter may occur (t[8; 14, 22 or 2] translocation),

19

resulting in c-myp,overexpressionu 'While it is not clear what
c-pyg;§_contribution.to the«development of the lymphoma is, it
apparently does not affect early B-progenitors’ ability to
further differentiate, as evidenced by 1) pre-B lines derived
from lymphomas of c-pyg transgenic mice can further rearrange
their Ig light chain loci (Adams et al., 1985) and 2) the
observation that infection of pristane-treated mice with a
recombinant retrovirus expressing' c-pyg, leads to a Ihigh
incidence of differentiated Ig-secreting plasmacytomas in mice
(Sawyers et al., 1991; Magrath, 1990). Apparently, to achieve
the fully transformed phenotype in Exp overexpressing cell
lines, it is necessary that another genetic event must occur
that affects normal differentiation, such as coexpression with
oncogenes such as v-Ha-pgp (Schwartz and Witte, 1988). The
above data suggests a role for pyg in cell proliferation.

While many'proto-oncogenes require some genetic mutation
for transforming activity (i.e. c—gfl is not oncogenic without
a 5’ -deletion and subsequent fusion to the LE; locus that
unmasks tyrosine kinase activity; c—pap apparently requires
specific point mutations that constitutively activate GTPase
activity [for review see Bishop, 1991] ) , it is clear that
simple over-expression of c-pyg, via juxtaposition to an Ig
promoter and within a temporal framework, is sufficient for
transforming-activity in EBV-immortalized cells (for review,
see Magrath, 1990). Therefore, the transforming mechanism of

myc should reflect the normal function of c-myc.

20

Kelly et a1. (1983) found that stimulators of the
proliferative response in B-lymphocytes (LPS, concanavalin.A)
and NIH/3T3 fibroblasts (PDGF) induce the expression of c-t_n_yg.
Coughlin et al. (1985) have shown that c-myg RNA is induced.by
EGF in human fibroblasts and that this signal is transduced
through PKC. Other functions, circumstantially ascribed to
EL: are repression of the non-overexpressed c-m allele
(Magrath, 1990) and entry into the cell cycle from a resting
state (Luscher and Eisenman, 1990). This data, coupled with
1135’s nuclear localization, sequence specific DNA binding
activity (Blackwell et al., 1990) and ability to
hererodimerize with ma; (Blackwood and Eisenman, 1990)
(reminiscent of the fps/ipp (AP—1) transcriptional activator
complex), suggests that c-myg is at the convergence of many
proliferation/signal transduction pathways.

myg’s seeming involvement in proliferation but not
differentiation has a notable exception. Murine
erythroleukemia cells (MEL cells) can be derived from splenic
lymphocytes after whole mouse infection with Friend
erythroleukemia 'virus. These immature erythroblasts can
almost fully differentiate (as assayed by induction of
hemoglobin synthesis) into erythrocytes by incubation with
dimethyl sulfoxide (DMSO). Coppola and Cole (1986)
transfected an LTR/c-myg construct into MEL cells and found
that its expression was correlated with inhibition of DMSO-

induced differentiation.

21

3.4 The ras Oncogene

The gap family of oncogenes have extensive homology to G
proteins, molecules found on the cytoplasmic surface of the
cell membrane and thought to be involved in the inward
transduction.of extra-cellular signals. ‘The current model for
the activity of G proteins is that they usually exist in the
inactive state, characterized by the binding of GDP,
associated with their upstream signalling molecule (probably
a receptor). When a stimulus is received from upstream, GDP
is exchanged for GTP, allowing the G protein to interact with
a downstream cytosolic effector molecule and resulting in
further propagation of the signal. Once this interaction is
accomplished, the GTP is hydrolyzed to GDP and the G protein
returns to the resting stateu It is though that pgp oncogenes
represent chronically-activated G proteins that continually
propagate growth or differentiation signals downstream“ This
chronic activation is conferred by point mutations in the c-
pgg gene allowing the production of oncogenic gag proteins.
The Egg oncogenes carried by the murine transforming viruses
v-K-pag and v-Har_a__s_ have Ser12 and Thr59 or Arg12 and Thr59
replacing Gly12 and Ala”, respectively, presumably leading to
chronic activation of these proteins (for review, see
Barbacid, 1987).

Recent work utilizing the cytoplasmic injection of
biotinylated anti-c-pap antibodies into B-cells, shows that

after crosslinking surface IgM, c-ras co-caps with these

22

molecules. This suggests that c-pgp is involved in the B—cell
receptor signal transduction pathway'(Graziadei.et.al., 1990).

c-pgp. protein clearly' has proliferative effects on
NIH/3T3 fibroblasts. Microinjection of anti-c-ppp antibodies
into the cytoplasm of these cells blocks serum stimulated Gy-
to-S phase entry but has no effect in cycling cells (Mulcahy
et al., 1985). Also, cells transformed with growth factor
receptors such as v—m, cannot enter S phase if microinjected
with anti-c-pgg antibodies (Smith et al., 1986). Overall,
however, it does not appear that over-expression of gpg is
sufficient in itself to fully transform cells.

Primary rat embryo cells transfected with the v-Ha-pas
oncogene may pass through a few cell divisions, but become
established as cell lines at a reduced frequency compared to
those also containing exogenously introduced c—myg (Barbacid,
1987). This suggests that another genetic transforming event
is needed for the fully transformed phenotype. This
correlates with. work. done 'with. murine jpre-B-cells which
suggests that while bone marrow cultures infected with v-Ha-
pap do establish cell lines in vitro, they require v-pyg co-
expression for the fully transformed phenotype (Schwartz et
al., 1986).

mg involvement in the differentiation programs of
lymphoid cells is fascinating in that lymphoid lineages can.be
transformed by v-Ha-gs but may or may not be blocked to
differentiation. The predominant outgrowth of high-density

bone marrow cultures infected with v-Ha-ras are pre—B-cells

23

that do not further differentiate in tissue culture. These
transformants proceed to clonality, have a high growth rate
and form tumors at a very low rate upon challenge in syngeneic
animals (Schwartz et al., 1986) . However, these transformants
do not take on the fully-transformed, tumor—forming phenotype
unless coexpressed with a nuclear oncogene such as v-myg
(Schwartz et al., 1986).

Examples of cell types that are transformed but not
blocked to differentiation are: JJ Erythroid progenitors
from spleens of phenylhydrazine—treated mice infected with ki—
pgs are able to undergo colony formation and differentiate
into hemoglobin synthesizing cells (Hawkins and Scolnick,
1981), 2) Spleen cells infected with v-Ha-pgp produce clonal
lines of mast cells that maintain the requirement of IL-3 for
growth (Rein et al., 1985), and 3) Infection of murine bone
marrow cultures with v-Ha-;g§_ yields two types of colonies in
soft agar: immature B-lymphoid progenitors and cells of the
myeloid phenotype (Pierce and Aaronson, 1985). ‘While the
characteristically-myeloid colonies could not be established
into cell lines, the more blastic-appearing monocytes could.
When the latter were treated with TPA, these blastic cells
acquired.myeloid markers and their cloning efficiency in soft
agar dropped. It is not clear whether these myeloblasts were
derived from B-lymphoid progenitors (so-called lineage
infidelity) or represented the true monocyte lineage.
However, Bretz et al. (1992) have found that clonal pre-B-cell

lines derived from v-Ha-ras-transformed high density bone

24
marrow cultures can further differentiate into functional

macrophages after syngeneic tumor challenge.

4. Possible Origins of CD5+ B-lymphocytes

With the knowledge that CD5+ B-lineage cells are
undetectable (by FACS analysis) in the bone marrow of mice
(see section 2.1), the following question presents itself:
Are the oncogenic viruses (particularly v—Ha—rfi) that give
rise to a preponderance of CD5+ B-lineage cell lines upon
infection of murine bone marrow (see section 2.4.2 above),
specifically targeting a rare population of CD5” 'B—l’ cells
and conferring a growth advantage via transformation, or are
the viral targets more diverse (including CDS’ ’B-2’ cells)
with the transforming event actually inducing the expression
of CD5? A body of evidence does exist that suggests the

latter may be possible.

4.1 The Modulation of CD5 Expression

Evidence that CD5 expression is not a static phenomenon
was first introduced by Miller and Gralow (1984) who were able
to induce CD5 expression on purified populations of human
peripheral CDS' B-cells after in vitro treatment with PMA, an
activator of protein kinase C (PKC). Since PMA has been
extensively used to induce activation or differentiation of B—
cells, CD5 was initially considered a marker for activation.
However, further evidence has shown that most CDS“ B-cells 1p

vivo are in GO or G1 and not activated (for review see Kipps,

 

25

1989). .Additional evidence that CD5 antigen. levels are
modulated was supplied by Jyonouchi et al. (1990) who found
that IL-4 down-regulated the CD5 levels on the 702/3 cells (a
murine pre-B-cell line) while LPS up-regulated.CD5 mRNA.levels
three to four-fold. Brooks et al. (1992) showed that CD5
transcript levels are modulated as BCLf4N13 cells (a clonal
murine B-cell line) traverse the cell cycle.

However, the most intriguing experiment demonstrating CD5
modulation was offered by Ying-zi et al. (1991). This group
used FACS to sort against the typical CD5+ B-cell and
collected a population of CD5'sIgM‘IgD1‘15""‘CD23*‘1‘3'hCD45high cells
(’B-2’ cells) from murine spleen. Using an anti-Mu antibody,
they then cross-linked surface IgM on this population and
noted an increase in the percentage of CD5-expressing cells,
as well as an increase in actual cell numbers upon resorting.
The authors claimed that if this increase had been due to the
expansion of a small population of contaminating CD5+ B-cells,
a bi-modal distribution would have been evident upon FACS
analysis. In their discussion, they hypothesized that there
were two types of activation pathways for ’B—2'-cells, one
that was LPS-inducible resulting in secreted Ig (standard B-
cell activation assay) with no increase in CD5, and the other
that proliferated and up-regulated CD5 expression upon anti-
sIgM crosslinking (but does not produce secreted Ig). In
addition, treating the CD5-inducible splenic populations with
interleukin-6, yielded a surface phenotype identical to

peritoneal-derived.CD5*B-cells. The authors interpreted.this

26
to mean that the CD5 antigen may be a marker for proliferation
of ’B-2' cells and that CD5+ B—cells may not represent a

distinct lineage.

1142 CD5’ B-cells Are Made. Not Born

The main pillar of support for the unique lineage
hypothesis comes from the work of Huang et al. (1986) (see
section 2.1) who found that, while donor bone marrow cells
were not able to reconstitute the CD5+ B-cell population in
lethally-irradiated mice, peritoneal-derived CD5+ B-cells
could. They interpreted this data to mean that CD5” B-cells
could only be derived from CD5+ precursors, effectively
establishing the CD5+ B-cell as a separate lineage. In
contrast, the work of Yin-zi et al. (1991), (detailed in
section 5.1) suggests that the CD5+ phenotype is available to
any immature (i.e. not previously activated) B—cell that
receives the appropriate stimulation. While neither of these
models directly addresses the literature detailing the high-
frequency of establishment of CD5+ B-lineage cell lines from
CD5‘ bone marrow by infection with transforming oncogenes
(section 3.2), that data is most reconcilable with the model
proposed by Yin-zi et al. (1991). If, as they propose, the
CD5+ phenotype is available to any immature B—cell, then a
signalling pathway must exist that is capable of up-regulating
CD5 expression after the appropriate stimulation. Since the
cellular homologs of transforming oncogenes are implicated in

the transduction of intracellular signals, it is formally

27
possible that the targets of viruses encoding these oncogenes
consist of both CD5+ and CD5' B-lineage cells. Therefore,
induction of CD5 expression in CD5’ B-cells may be the result
of oncoprotein-mediated chronic activation of signalling
pathways involved in CD5 up-regulation.

While the work of Yin-zi et al. (1991) is very solid, one
technical objection may be raised. The authors initially
collected their CDS' B-cell pools from heterogeneous B—cell
populations that presumably included CDS" B—cells. It is
formally possible, although unlikely, that what they
interpreted as an induction of CD5 expression on CD5' B-cells,
could actually have been the expansion of a contaminating
population of CD5+ B-cells. While FACS analysis did not
detect the bi-modal distribution one would expect from the
expansion of a single population in a pool of two cell types,
bi-modal peaks may have been present but obscured by
background (or too close together to discriminate). The use
of clonal lines of CD5‘ B—lineage cells would obviate this

objection.

4.3 .A Thesis Proposal

 

This thesis addresses the hypothesis that expression of
the CD5 surface antigen on B-lineage cells can be induced by
the introduction of transforming retroviruses and their
associated oncogenes. This work relies upon CDS’ clonal B—
lineage cell lines to ensure that the retroviruses or their

associated oncogenes are not conferring a growth advantage to

28
a minor contaminating population of CD5+ cells, whose
subsequent expansion could possibly mimic CD5 induction.
Additionally, the putative CD5 promoter has been cloned in
order to initiate work on a possible mechanism governing the

expression of CD5.

29
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34

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35

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36

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CHAPTER 2

Cloning and Tissue-Specific

Regulation of the Murine CD5 Promoter

Timothy R. Weichert and Richard C. Schwartz

Department of Microbiology, Michigan State University

East Lansing, MI 48824-1101

37

38
ABSTRACT
A genomic clone containing 1700 base pairs of the 5’-flanking
region and first exon of the murine CD5 gene was isolated by
screening an NIH 3T3 fibroblast genomic library with the
previously characterized murine CD5 cDNA (Huang et al. (1987)
PNAS 84, 204-208). The CD5 5'-flanking region lacks a
consensus TATA box but contains the reverse IgH uEZ, reverse
CCAAT, AP-l and TCF—2a motifs. Transcription is initiated
from multiple sites upstream of an INR-like sequence. When
linked to the CAT reporter gene, the CD5 5'—flanking region
was an active promoter in transient transfection assays using
the EL4 (T—cell), PD36 (pre-B-cell) and M12 (B-cell) lines,
but was inactive in NIH 3T3 fibroblasts. This pattern of
lymphoid-specific expression reflects the pattern of in vivo
CD5 expression. Successive 5’-to-3’ deletions of the CD5
promoter/CAT reporter were transfected into T and B-cells
revealing a succession of positive and negative regulatory
elements until promoter activity was eliminated at position -
27. This result identified sequences from -125 to +9, (which
contain the INR, reverse IgH pE2 and AP-l sites) as a minimal
promoter. Isolation and characterization of the CD5 promoter
represents an initial step in elucidating the control over

tissue-specific expression of the murine CD5 gene.

39
INTRODUCTION

The murine CD5 surface antigen (also referred to as Ly-l
or Lyt-l) is a 67 kDa MW monomeric, membrane-associated
glycoprotein that is structurally homologous to the cysteine-
rich scavenger receptor family of polyanion-binding receptors
(1,2). It is lymphoid-specific in its expression, being found
on all T-lymphocytes and on a subset of B-cells found mainly
in serousal cavities of adult mice. CD5 is physically
associated with the TCR/CD3 complex on T—cells where it is
thought to be a jphosphorylation target for the protein
tyrosine kinases p561Ck and p59fyn during the transduction of
extracellular signals (3,4). No such function has been
attributed to B-cell-associated CD5, but the B-lymphocyte-
specific surface antigen CD72 (Lyb-2) has been identified as
a ligand for CD5 (5). The implications of CD72/CD5 binding
for T-B and B-B-cell interactions have yet to be elucidated.

Some investigators consider CD5‘IB-cells to be a unique
lineage of B-lymphocytes, the so-called B-1 cells (B—2 cells
do :not express CD5)(6). This idea stems from .adoptive
transfer experiments that showed that peritoneal-derived B-
lymphocytes were capable of reconstituting CD5+ B-cells in
lethally irradiated mice, while bone marrow-derived CDSTHflf
B-2 progenitors could not (7). However, other groups have
found that CD5 expression on B-cells may not be the static
phenomenon.predicted.by'a lineage model and that levels of CD5
expression may be regulated. It has been found that anti—

immunoglobulin (Ig)-crosslinking of surface IgM on CD5‘ B-

40

cells induces the expression of CD5, and acts as a mitogen
(8). Additionally, CD5 expressed on clonal murine B—cell
lines has been found to be down-regulated by
lipopolysaccharide (LPS) and modulated as the cell traverses
the cell cycle (9,10). CD5 expressed on human peritoneal B-
cells has been found to be up-regulated in the presence of PMA
(11). In either case, the lymphoid specificity of CD5
expression, together with its restriction to a B—cell subset
and possible regulation in response to signal transduction
events, suggests complex regulatory mechanisms may govern the
expression of CD5.

To elucidate the mechanisms regulating the expression of
the murine CD5 gene, we have cloned the murine CD5 promoter
from an NIH 3T3 fibroblast genomic library. The CD5 5’-
flanking region lacks a TATA box and transcription of the CD5
message is initiated from multiple sites flanking an INR-like
sequence. The CD5 promoter is capable of a pattern of tissue—
specific expression reflective of ip_yiyp CD5 expression and
is(governed.by'both.positive and.megative regulatory elements.
The promoter sequence contains consensus TCF-2a and IgH pEZ
motifs, both of which have been shown to be involved in the
regulation of tissue-specific expression from.other lymphoid—
specific promoters (12,13). Deletion of promoter sequences
from -127 to -25 (which contain AP-l, SV40 core enhancer and
the initiator [INR] motifs) results in loss of promoter
activity, This region will be useful in elucidating

reEnlatory mechanisms governing expression of the CD5 gene.

41
MATERIALS AND METHODS

Cell Culture and Transfection Assays - Murine EL4 T—cell (ATCC
TIB 39), M12 B-cell (14) and PD36 pre-B-cell (12) lines were
maintained in Rosewell Park Memorial Institute (RPMI) medium
1640 supplemented with 10% fetal calf serum (FCS), SOpM 2-
mercaptoethanol (2ME) and 25pg/mL each of penicillin and
streptomycin (penn/strep). NIH 3T3 murine fibroblast cells
were maintained in Dulbecco’s Modified Eagle’s Media (DMEM)
supplemented with 10% calf serum and pen/strep as above. The
lymphoid lines were transfected by electroporation (Biorad
Gene Pulser, Biorad, Inc., Hercules, CA) utilizing parameters
of 280 volts and 960 uF capacitance conducted.across a cuvette
of 0.4 cm electrode gap (Biorad). Pulse times varied between
25 and 35 msec. Each cuvette contained 0.5 ml RPMI media/10%
FCS (without 2ME or penn/strep) with 107 cells, 4.4 pg pGL2
luciferase reporter plasmid (Stratagene, Inc., La Jolla, CA)
as an internal control (Fig. 4d) and 10.0 pg of the
appropriate CAT reporter* construct (Figure 4a).
Electroporated cells were transferred from cuvettes to 6 cm
tissue culture plates in 5.0 ml complete medium. NIH 3T3
cells were transfected using quantities of plasmid identical
to the lymphoid lines but utilizing the calcium phosphate
coprecipitation method (15).

After 24 hours, transfected.cellS‘were washed.in ice-cold
phosphate-buffered saline and divided into a 1/5th portion for
luciferase assays and a 4/5th portion for CAT assays. The

cells analyzed for luciferase assays were lysed in 50.0 pl

42
cell culture lysis buffer (Promega, Inc., Madison, WI) and
20.0 pl used for luminometry in a Turner TD-20e luminometer
(Turner Designs, Sunnyvale, CA) utilizing 100.0 pl luciferin
assay buffer (Promega). Light unit readings were collected
for 15 seconds (after a delay time of 30 seconds) and the
ratio of each sample value to the lowest sample value
determined (normalized luciferase ratios). Normalized
luciferase ratios were used to determine the volume of cell
extract used in the CAT reaction (16). Cell extracts for CAT
assays were prepared by freeze—thawing cells in 0.25 M Tris-
HCl for 3 cycles, then heating to 65°C for 20 mins. CAT
reactions were carried out utilizing 1.0 pl of 50.0 mCi/mmol
[“CJChloramphenicol, 25.0 pg of n—butyryl Coenzyme A, with
cell extract and 0.25 M Tris-HCl in a total volume of 125.0
pl, and incubated at 37°C for 10 hours. Conversion of
[“C]chloramphenicol to butyrylated forms was directly
determined from TLC plates on an Ambis Radioanalytic Imaging

System (Ambis, San Diego, CA).

Cloning Strategy and Sequencing - An NIH 3T3 fibroblast
genomic library, generated by BamHI partial digest (15 kb
average insert size) and cloned into the XhoI sites of the
Lambda Fix II vector (Stratagene), was probed with the NCOI-
EcoRV restriction fragment of the murine CD5 cDNA clone (Fig.
1a)(17). 11F plaques were screened resulting in the isolation
of six hybridizing clones. ‘Two of these clones were analysed,

with one (LambdaFixIIcl.41-CD5) ultimately yielding the 5’-

43

Figure 1. Cloning Strategy and Partial Restriction Map of the
CD5 5’-Flanking Region. A. Partial restiction map of the CD5
cDNA (17) . The NcoI-EcoRV restriction fragment was used as a
probe to isolate LambdaFixIIcl.41-CD5 from an NIH 3T3
fibroblast genomic library. Also, the 263 bp BamHI-BstEII
fragment of the CD5 cDNA was ligated in the antisense
orientation into pSP64 to create the pSP640-263 in vitro
transcription vector (panel B). B. Partial restriction map
of LambdaFixIIcl.41-CD5 genomic clone. This clone was
digested with SacI and fragments ligated into the
corresponding site in SK-pBluescript (Stratagene) (panel C).
C. Restriction map of pBlu.41. Nylon transfer-membrane
colony lifts of pBluescript transformants were probed with the
NcoI-EcoRV fragment of the murine CD5 cDNA (panel A) to
identify pBlu.41. Arrow below panel represents direction of
double-stranded sequencing using a primer complementary to the
pBluescript vector. D. Restriction map of M13mp19proCD5.
LambdaFixIIcl.41-CD5 (panel B) was digested with NooI,
yielding a 2.0 kbp fragment. This fragment was digested with
Sphl and directionally subcloned into the Sphl and PstI sites
of M13mp19 to create M13mp19proCD5. Arrows below figure
represent direction of single-stranded sequencing using an
M13mp19-specific primer and a second primer complementary to
insert sequences. E. Restriction map of the in vitro
transcription vector pSP640-263pro. The NcoI-StuI restriction
fragment of M13mp19proCD5 was subcloned into the NcoI and SmaI
sites of pSP640-263 (panel A).

Abbreviations: A, AvaI; Acc, Ach; B, BamHI; dB, destroyed
BamHI site; Bx, BstXI; B2, BstEII; E1, ECORI; E5, EcoRV; H,
HincII; N, NcoI; S, SacI; Sa, Sau96I; Sph, SphI; St, StuI; V,
vector sequences; M13, M13mp19 vector sequences; Blu,
pBluescript vector sequences.

44

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45
flanking region (Fig. 1b). 'Restriction fragments from.a SacI
digest of LambdaFixIIcl.41-CD5 were subcloned into the SK-
pBluescript plasmid and rehybridized.with the.NCo1-EcoRV cDNA
probe to select a positive clone (pBlu.41) with a 500 bp
insert (Fig. 1c). Double-stranded, dideoxy-sequencing
(Sequenase version 2.0; USB Inc., Cleveland, OH) utilizing the
pBluescript reverse primer (5’-AACAGCTATGACCATG-3’;
Stratagene)(18) determined.that this fragment terminated.29 bp
upstream of the coding region. The 500 bp fragment was then
used as a probe to identify a genomic clone containing a 2.0
kb NboI genomic clone fragment, which was made blunt-ended
with.Klenow fragment of DNA polymerase I (Klenow) and digested
with SphI. This yielded a 1.7 kb fragment that was
directionally subcloned into the PstI (blunt-ended with T4 DNA
polymerase) and SphI sites of M13mp19, creating M13mp19pro
(Fig. 1d). This effectively regenerated the 3’.NCoI site of
the genomic clone. Approximately 1.0 kb of this insert was
sequenced utilizing the M13 forward sequencing primer (5’-
TGACCGGCAGCAAAATG-3’)(USB) and an upstream primer (5’-
CCTGCTCACTCCTGATG-3’) complementary to the insert sequence at

-496 to —512.

Plasmid Constructs - To create the in vitro transcription
vector pSP64CD5 for RNAse protection assays, the murine CD5
cDNA was digested from the MD—10 vector (17) by BamHI (Fig.
1a), cloned into the corresponding site in the pSP64 vector

(Promega) and then antisense orientation was verified by

46

multiple restriction. enzyme digestions. 'This construct,
pSP64CD5, was then digested with BstEII (restriction site
found at +263 of the CD5 cDNA) and HincII (this site is found
in the pSP64 polylinker between the SP6 promoter and the CD5
insert). The resulting restriction fragments were subjected
to low-melting-point-agarose (LMP) gel electrophoresis and the
band containing the vector sequences and the first 263 base
pairs (bp) of the CD5 cDNA.was isolated from the agarose. The
vector was made blunt-ended. with KlenOW’ and the vector
recircularized with T4 DNA ligase, creating pSP640-263.
pSP640-263 was then digested.with.NCoI and SmaI (the SmaI site
is found in the pSP64 polylinker upstream of the cDNA insert)
and the .NCoI-StuI 5'-flanking region fragment (Fig. 1d)
ligated into it to generate pSp640-263pro (Figure 1e).

The 1700 bp.NCoI (blunt-ended with Klenow)-SphI fragment
frtm1M13mp19CD5 (Fig; 1d) was directionally subcloned.into the
PstI (made blunt-ended.by T4 DNA.polymerase) and SphI sites of
the pCAT-Basic reporter vector (Promega) (Fig. 4a and 4b).
This 'vector' was digested. with .NCoI (and .HindIII and. the
resulting 1700 bp fragment subcloned into the corresponding
sites in the pCAT-Enhancer vector (Promega) (Fig. 4c). The
-125 deletion constructs were created by directly ligating a
purified Sau96I-NCoI (both blunt-ended with Klenow) fragment
of the 1700 bp M13mp19CD5 insert into the PstI (made blunt-
ended with T4 DNA polymerase)-HindIII (made blunt-ended with
Klenow) sites of pCAT-Basic and pCAT-Enhancer vectors.

Correct orientation was verified by multiple restriction

 

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47
enzyme digestions. Sequential 5'—to-3’ deletions of
promoter/reporter constructs were accomplished by utilizing
the HindIII site of the reporter vector and the appropriate

internal restriction sites (Fig. 4a an 4d).

RNA Isolation and Characterization - Cytoplasmic RNA was
isolated from EL4 and NIH 3T3 cells by an SDS/urea method (19)
and analysed by RNAse protection assay (20). Riboprobes were
synthesized in vitro in the presence of 10 units of SP6 (New
England Biolabs (NEB), Beverly, MA), 1x SP6 transcription
buffer (NEB), 6.0 pM of 400 Ci/mmol [a-”P]CTP, 150.0 pM each
of unlabeled ATP, UTP and GTP, 25.0 pM unlabeled CTP and 1.0
ug linearized vector (Fig. 1e). Probes were hybridized with
180.0 pg cytoplasmic RNA in 80% formamide, 40.0 mM PIPES pH
6.7, 0.4 M NaCl and 1.0 mM EDTA overnight at 50° CL
Riboprobe-cytoplasmic RNA hybrids were digested in 10.0 mM
Tris-HCl, pH 7.5, 5.0 mM EDTA, 300.0 mM NaCl, 2.0 pg/ml RNAse
T1 and 40.0 pg/ml RNAse A at 30°C3for 30 minutes. Protected
RNA species were electrophoresed in a 0.4 cm-thick 5.0%

polyacrylamide/urea sequencing gel (19).

RESULTS
Sequence of the CDS 5’—Flanking Region and First Exon -
Sequence analysis of pBlu.41 (Figure 2) reveals that an intron
begins at +58 bp downstream. of the terminal nucleotide
reported as the 5’—end of the murine CD5 cDNA (17). Our data

shows a single nucleotide discrepancy in this 58 bp coding

48

Figure 2. Sequence of the CD5 5’—Flanking Region. The
sequence from -27 to +87 was derived from pBlu.41 and the
sequence from +9 to -1119 was derived from M13mp19proCD5.
Numbers preceded by ’-’ at the right margin refer to bp
upstream of a transcriptional initiation site at +1. Location
of known enhancer motifs are highlighted by overlining or
underlining. The N-terminal methionine of the coding sequence
is identified as ’met’ . A guanine nucleotide not matching the
reported cDNA sequence (17) is identified by a solid box.

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49

T89998T898
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9898989898
8TT8998T89
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GCTTGTGTDT
T8888T8898
T88T8888TT
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GRCRCCTGGT

TT88988898
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RTTGGGGDRG

9888TT8889

RGRGRTDRCC
889T888988
RTGRRTGRRT
RTCCRGRGRC
CTCRTRCCTR
GRGRTDTCTC
9898989898
89T8998989
TTCTTRGGCR
9889889889
TDTTGTTTTG
RTTRTGGRCT
TGBTTTTCDT
98T8888988
888TTTT988
TT8889888T
TCTGDDCRGD

TT888T8T8T

TGCRTT8T98'4019

GRRTGRRTGR

89T989989T-919

RDRTGGRGRT

T8T8T8T818-819

9898989898

8T8T88T888'719

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T88988888T-519

TRTTGRCDCT
8898T89899
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+1
7898889889 89988889TTRT88 918 898 188 898 899 8T8 818 +27
. met intron ....................
818 881 888 988 198 818 8T8 889 988 8T8 48T898T8T8T888

88T98T888T98T8889

FIGURE 2. Sequence Analysis of the CD5 5’-Flanking Region

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50

region when compared to the reported cDNA sequence.

Originally reported as adenine (17), we find. a guanine
nucleotide at base +39. While this A to G alteration may
represent a polymorphism between the C57Bl/6 strain used to
isolate the CD5 cDNA and the Swiss mouse strain (NIH 3T3) used
to clone the 5'-flanking region, the RNAse protection data
(Figure 3) in this area does not display truncated RNA
species, suggesting that the base is indeed guanine. In
either case, the A-to-G change is silent, with both resulting

triplets coding for threonine.

The 5’ End of the CD5.Message.Maps to the 5’-Flankinngegion —
To determine if the 5’-end of the CD5 transcript actually
mapped to our 5’—flanking region genomic clone and to
determine the transcriptional start site of the mRNA, a hybrid
gene was constructed in the pSP64 vector (Figure 1e). The
StuI-NCoI fragment of the CD5 5’—flanking region was
directionally cloned into the pSp640-263 vector already
containing the terminal 263 bp fragment of the CD5 cDNA. This
vector, pSp640-263pro, maintains the fidelity of the NcoI
coding region-junction area, without additions or deletions,
and produces a riboprobe (run-off the SP6 promoter)
complementary to the 5’-end of the CD5 coding region that
extends into the flanking region.

Figure 3 shows an RNase protection assay of EL4 T—cell
cytoplasmic RNA compared to fibroblast RNA (CD5’ cell type)

using the riboprobes generated from the pSp640-263pro vector.

51

FIGURE 3. The 5’-Termini of CD5 mRNA Map to the CD5 5’-
Flanking Region. IRNAse protection analysis of CD5 mRNA. Lane
2, NIH 3T3 fibroblast RNA probed with probe P. Lane 2, EL4
RNA probed with probe P’. Lane 3, EL4 RNA probed with probe
P. Lane M, single—stranded RNA ladder (BRL, Gaithersburg, MD)
end-labelled with T4 polynucleotide kinase and [7—32P]UTP.
Size of marker bands is given in nucleotides on right border
of figure. Lane P, probe P. This probe was generated by ip
vitro transcription utilizing the EcoRI-linearized pSP640-
263proCD5 vector (Figure 1e). Full-length probe P is 516
bases, of which 471 are CD5-specific (263 bases complementary
to CD5 cDNA.and 208 bases complementary to the CD5 5’-flanking
region) while 45 bases are encoded by the pSP64 polylinker.
Lane P’, probe P’. This probe was generated by in vitro
transcription reaction utilizing the NooI—linearized pSP640-
263proCD5 vector (Figure 1e). Full-length probe P’ is 290
bases, of which 263 bases are complementary to the CD5 cDNA
sequence and 27 bases are pSP64 polylinker sequence.
Arrowheads without tails at the left border of the figure
identify multiple protected species of EL4 RNA.in lane 3. 'The
arrowhead with a tail at the left border identifies a single
protected species of EL4 RNA in lane 2.

FIG

52

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FIGURE 3. The 5’-Termini of CD5 mRNA Map to the CD5 5’-
Flanking Region

1')

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53

EL4 RNA (Figure 3, lane 3) displays protected species

of heterogeneous length terminating in the putative promoter
region of pSp640-263proCD5. No protected species are evident
with fibroblast RNA (Figure 3, lane 1). pSp640-263 control
protections, utilizing probe P’ (complementary to the CD5
coding region only) displays a single protected band
terminating at the 5’-NCoI site of the coding region, while
most protected RNA species using probe P (which includes the
5’-flanking region) terminate Lm>tx> 100 bases beyond this
point. This suggests that CD5 transcription initiates from
multiple sites in the 5’-flanking region.

The reported cDNA sequence reveals only 6 nucleotides
upstream of the ATG coding for the N-terminal methionine of
the leader sequence (17). Due to the apparent heterogeneity
of start sites, the cDNA sequence may have been derived from
an mRNA species initiated at a site close to the coding
sequence. Therefore, downstream nucleotide numbering will
commence with the 5’-terminal thymidine (T) of the published
cDNA sequence (17). SI nuclease protections utilizing both
synthetic oligonucleotide probes and single and double-
stranded restriction fragments, as well as primer extension
analyses with multiple oligos, were unable to map the 5’-
termini of CD5 transcripts with any finer detail, possibly due

to secondary structure of the flanking region.

The CD5 5’-Flanking Region Lacks a Consensus TATA Box - The

5’-flanking region of CD5 lacks a consensus TATA box (Figure

54
2). Two general types of TATA-less lymphoid-specific
promoters have been described. The first, represented by the
TdT promoter, uses the initiator (INR) sequence, 5’-
CTCANTNGGAGAC-3’, to initiate transcriptitnlfrtmla single site
in the INR sequence. The group represented by Ig variable
region.promoters expressed.in.pre-B—cells, and the mb-l and.B—
29 genes, also contains this INR sequence but initiates
transcription from multiple sites upstream of the INR (21,22) .
The 5’-flanking region of CD5 has an INR-like sequence (5’-
CTCACTGAQCTG—B’; underlined bases match the INR sequence) at
-29, overlying a putative SP1 binding site (Figure 2). In the
mb-l promoter, SP1 is thought to act as a tethering factor for
TFIID and the transcription complex (21). Our clone probably
belongs to the mb-l class of TATA-less promoters although the
multiple initiation sites of CD5 appear to range further

upstream than those described for this type of promoter.

The CD5 5’-Flanking Region is Capable of Tissue-Specific
Promoter Activity - CD5 mRNA is expressed in a lymphoid-
specific fashion in mice and humans (17). To investigate
whether our putative promoter could initiate transcription in
a lymphoid-specific manner at levels reflective of those found
in vivo in T-cells and B-cells (T-cell steady—state mRNA
levels are consistently greater than B-lineage levels; FACS
analysis of surface CD5 protein also reflects this
difference), we utilized transient transfection techniques to

introduce the -1700 pCAT-Basic reporter vector construct

55

FIGURE 4. Restriction Map of the CD5 5’-Flanking Region
Deletion Constructs. A. The —1700 bp CD5 5’-flanking region
fragment was subjected to serial 5’-to-3’ deletions with Ach
(-1107), AvaI (-374), BstXI (—275), StuI (-208), HincII (-

165), Sau96I (-125) and SacI (-27). Numbering of deletion
constructs corresponds to the restriction site used to
generate them . The maps of -275 through -27 have been

expanded to show greater detail. Location of known enhancer
motifs are identified above each construct beginning with -
275. B. Restriction map of pCAT-Bacic vector (Promega). C.
Restriction map of pCAT-Enhancer vector (Promega). D.
Restriction map of the pGL2—Luciferase control vector

(Promega).

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Deletion Constructs

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57
(Figure 4a) into EL4 (T—cell), M12 (B—cell), PD36 (pre-B—cell)
and NIH 3T3 (fibroblast) cell lines. Figure 5 shows that
promoter/CATIactivity'in‘these cell types is indeed reflective
of relative steady state mRNA levels found in these cell
types. EL4 T-cells displayed the greatest activity, followed
by PD36 pre-B-cells (0.8x the value of EL4) and M12 B—cells
(0.5x the value of EL4), with NIH 3T3 fibroblasts displaying
negligible CAT activity. CAT activity from the pCAT-Basic
vector alone was at background levels, while expression from
the vector containing the SV40 promoter and enhancer regions
(a. positive control for expression) showed the greatest
activity in any given line. If the orientation of the CD5
promoter is reversed in the pCAT-Basic vector, expression

decreases 10-fold (Figure 6).

Sequences Flanking —27 to —125 are capable of Acting as a.
Minimal Lymphoid-Specific Promoter - Figure 4a shows the
structure of pmogressive 5’—to-3’ CD5 promoter truncations
constructed in the -1700 pCAT-Basic and -1700 pCAT-Enhancer
vectors. When these constructs were transiently transfected
into EL4 cells, it was found that the vector containing the
-27 to +9 fragment was incapable of expression in either
plasmid, while the -125 to -27 fragment contains sequences
sufficient for" promotion (Figure 7). Putative enhancer
elements located.in this fragment (Figure 4a) that are capable
of transcriptional activation.inIother systems, include an AP-

1 site at -63 (23), a reverse IgH chain pE2 enhancer motif at

58

FIGURE 5. Tissue-Specific Expression of the CD5 Promoter.
Cell lines were transfected as described in Materials and
Methods. PD36 cells are poorly transfectable, with the pCAT-
Basic vector transfectants displaying only 1.5 light units of
activity. All cell extracts volumes were normalized to this
value for CAT reactions. After chromatography, TLC plates (PE
SIL G/UV; Whatman, Inc., Hillsboro, OR) were sprayed with
surface autoradiography enhancer (EN3Hance; NEN, Boston, MA)
and exposed for 48 hours. Numbers immediately below figure
represent the ratio of [14C] chloramphenicol converted by a
given transfectant to [14C] chloramphenicol converted by the EL4
-1700 transfectant. Figures are representative of at least
two replications per cell line.

Abbreviations: -1700, —1700 pCAT-Basic vector (Figure 4a); V,
pCAT-Basic vector; ’+’ , pCAT-positive control vector (contains
the SV40 promoter and enhancer regions; Promega).

59

EL4 (T) PD36 (pre'B)

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—1700 V + —1700 V +
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FIGURE 5. Tissue-Specific Expression of the CD5 Promoter

60

FIGURE 6. Maximal CD5 Promoter Activity is Orientation-
Dependent. The —1700 promoter fragment (Figure 4) was cloned
into the pCAT-Basic vector in the reverse orientation. The

-1700revu pCAT-Basit:construct was transiently'transfected, in
parallel with the -1700 pCAT-Basic vector, into EL4 cells.
The pCAT-positive control vector had the the lowest light unit
readings (2.0) and volumes of cell extracts utilized for CAT
reactions were normalized.to this numberu INumbers immediately
below the figure represent the ratio of [“C]chloramphenicol
converted by a given transfectant to [14C] chloramphenicol

converted. by" the -1700 pCAT-Basic transfectant. Figure
represents data from one experiment.

Abbreviations: ’+’, pCAT-positive control vector; -1700,
-1700 pCAT-Basic in the forward orientation (Figure 4);
-1700rev., -1700 pCAT-Basic in the reverse orientation; V,

pCAT—Basic vector only.

PIGU

61

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FIGURE 6.

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Maximal CD5 Promoter Activity is Orientation-
Dependent

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62
-39 (13) and a reverse SV40 core element at —109 (24). The
SacI site ‘used. to (generate the ~27 deletion. destroys a
putative INR site and. an overlying' SP1 motif, possibly
accounting for the loss of activity.

The CD5 promoter contains a reverse IgH chain enhancer
element, pE2, at -39 (5’-GCCAGCTG-3’). ;nr2 is capable of
enhancing expression from minimal promoters in both lymphoid
and non-lymphoid cells (13). pE2 is found in the ENHfi,intron
of the IgH heavy chain locus, situated between the pES and the
pE3 enhancer motifs. Studies utilizing a CAT expression
vector construct consisting of only a TATA box and the pE2,
pE3 or pE5 enhancers in various combinations, have shown that
pE2 is not required for tissue—specific expression of this
locus (25). While pE3 alone is active in all tissue types,
it’s non-lymphoid expression is suppressed by pE5. Constructs
consisting of pE5 and pE2 in tandem do not enhance expression
in any cell type. While this work implicates pES as the
genetic switch determining tissue-specific expression, it does
not address the ability of pE2 to enhance expression in the
presence of other cis—acting elements. The CD5 promoter may
be a good candidate for studying the capability of pE2 to act
as an enhancer outside the IgH locus.

The py tract (8-to-14 pyrimidine base pairs) of Ig
enhancers is found up to 41 bp upstream of heptamer/octamer
motifs and has been found to augment transcriptional
enhancement from these motifs. Deletion of the py sequences

have been shown to reduce promoter activity 2-3 fold in B-

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63
cells (26). CD5 has a 12 bp pyrimidine tract at —90 (5’-
CCCCCTCCCTCC-3’) which ends 40 bp upstream of the reverse pE2
motif. While the effect of py tracts on enhancement by pE
motifs is not known, the CD5 promoter may' provide the

opportunity to study such an interaction.

Regulation of the CD5 Promoter in Lymphoid Cells is.Mediated
by Positive and Negative Elements - Serial 5’ -deletions of the
CD5 promoter region reveal a succession of positive and
negative regulatory' elements in. both EL4 and. M12 cells
(Figures 7 and 8). Expression from. individual promoter
deletion constructs, in both EL4 and M12 cells, appear to
follow similar trends. Although [“C]chloramphenicol
conversion values are higher with deletion constructs
expressed in M12 cells, corrected values accounting for the 5-
fold less extract loaded in EL4 CAT assays suggest that
mutants express comparably in both cell lines.

The -275 and -125 promoter deletions in EL4 cells (Figure
7, upper panel) each display >2—fold increases in expression
over the preceding construction and may represent loss of
silencer elements from the promoter. The largest decrease in
expression compared to a preceding construction, is found in
the —165 deletion transfected into EL4 cells (14 fold
decrease) and may represent loss of an important enhancer
element found.between.positions -208 and -165 (Figure 7, upper
panel). Interestingly, the -165 deletion construct also

displayed.a drop in expression.in.M12 cells (3-fold; Figure 8,

64

FIGURE 7. .Activity of C05 Promoter Deletion Constructs in EL4
Cells. The deletion constructs depicted in Figure 4a were
transfected into EL4 cells with the -208 pCAT-Enhancer
construct yielding the lowest light unit values (4.5). All
cell extract volumes were normalized to this value for CAT
reactions. Numbers along the lower border of the figure
correspond to the promoter deletion construct (Figure 4a)
cloned into either the pCAT-Basic vector (upper panel) or the
pCAT-Enhancer vector (lower panel). Numbers along the upper
border of the upper panel represent the ratio of
[14C] chloramphenicol converted by a given transfectant to
[“C]chloramphenicol converted in -1700 pCAT-Basic cell
extracts and apply to the 'upper panel only. Data is
representative of three replications of this experiment.
Abbreviations: B, pCAT-Basic vector; E, pCAT-Enhancer vector;
’+’, pCAT-positive control vector.

1.8 0.7 1.9 7.0 0.5 1.6 <1 <.1

1.0

65

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66

FIGURE 8. .Activity of CD5 Promoter Deletion Constructs in.M12
Cells. The deletion constructs depicted in Figure 4a were
transiently transfected into M12 cells. The pCAT-postitive
control vector displayed.the lowest light unit readings (23.6)
and loading of cell extracts for CAT reactions were normalized
to this value. Numbers along the lower border of the figure
correspond to the deletion constructs (Figure 4a) constructed
in either pCAT-Basic (upper panel) or pCAT-Enhancer vectors
(lower panel). Numbers along the upper border of the upper
panel represent the ratio of [“C]choramphenicol converted by
a given construct to [“C]chloramphenicol converted by the
-1700 pCAT-Basic construct and apply to the upper panel only.
Data is derived from one experiment.

Abbreviations: B, pCAT-Basic vector; E, pCAT-Enhancer vector;
’+’, pCAT-positive control vector.

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68
upper panel), suggesting that T-cells and.B-cells have common
factors that recognize an enhancer element between positions
-165 and -208. Known enhancer elements found in this region
(Figure 4a) include a reverse CCAAT box (-198), thought to
bind the C/EBP family of transcriptional activators (27), and
a PEA3/ets motif (-174) (28), and a TCF-Zd motif (13).

The CD5 promoter contains a reverse CCAAT box motif at
-198. This motif has been shown to bind a diverse family of
proteins capable of transcriptional enhancement including
IgEBP-l (29), C/EBP (27) and.NF-IL6 (30). IgEBP-l is found in
all tissues although levels are highest in EL4 Técells and
pre-B-cells (29). NF-IL6 can be found in most tissue types
(30), while C/EBP is found in liver, adipose tissue, small
intestine, placenta, lung and in non-dividing tissue (27).
C/EBP, and other C/EBP—related proteins (CRP’s) are able to
heterodimerize, possibly' conferring' tissue specificity' or
gradations of enhancement to promoters containing this motif.
The CRP core motif is 5’-TT/GNNGNAAT/G-3’ with further
specificity conferred by flanking sequences. The CD5 promoter
contains three degenerate CRP motif homologies: one in
reverse orientation at -198 (which includes the reverse CCAAT
box) and two in standard orientation at -177 and ~234. The
sites at -177 and -198 deviate one base from the consensus
sequence, while the site at -234 is a perfect match. The
ubiquitous expression of the C/EBP family and ability to
enhance transcription from degenerate motifs makes the above

sites jpossible candidates for'Ibinding' of ‘transcriptional

1'19
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by,

a ‘.‘

69
activators in the CD5 locus. The redundancy of these sites
may be responsible for the lack of effect observed with the
deletion that removes only the upstream -234 CRP site.

The CD5 promoter contains TCF-2d motifs at -175 and -231
(Figure 2) . This domain, in the TCR-oz promoter, has been
shown to bind the lymphoid-specific factor LEF-l in nuclear
extracts from the 1200M T-cell line (13). This motif was
capable of enhancing transcription from heterologous promoters
in M12 B-cells, PD36 pre-B-cells and EL4 T—cells.
Transcription was minimal in NIH 3T3 cells, suggesting that
this factor is capable of conferring tissue-specific activity
(13). While this motif overlaps the PEA3/ets motifs at ~175
and -231, it would represent an ideal candidate for initial
investigation of factors conferring tissue-specificity to the
CD5 promoter. Again, the redundancy of these sites may
explain the lack of effect with the deletion that removes only

the upstream -231 TCF-2d site.

Sequences Upstream of -275 May Repress Expression of the CD5
Promoter in Fibroblasts — Figure 9 displays CAT expression
from pCAT—Basic vector deletion constructs transfected into
NIH 3T3 fibroblasts. In order to observe low levels of
expression, CAT reactions were loaded with 15-fold more cell
extract than used for the NIH 3T3 CAT reactions displayed in
Figure 5. The deletion constructs -1700, -1107 and -374
display equal but low-level promoter activityn This activity

increases 3.5—to-5-fold.for deletion constructs -275 to —125.

70

FIGURE 9. .Activity'of CD5 Promoter Deletion Constructs in.NIH
3T3 Fibroblasts. The deletion constructs in Figure 4a were
transfected into NIH 3T3 fibroblasts. The -125 pCAT-Basic
construct displayed the lowest light unit levels (21.0) and
all cell extract volumes for CAT assays were normalized to
this value. Numbers along the lower border of the figure
correspond to the deletion constructs in Figure 4a. Numbers
along the upper border of the figure represent the ratio of
[“C1chloramphenicol converted by a given cell extract to the
[“CJChloramphenicol converted by the -1700 pCAT-Basic cell
extract. The pCAT-positive control transfection is found in
the NIH 3T3 panel in Figure 5. Data is derived from one
experiment.

Abbreviations: V, pCAT-Basic vector.

71

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72
This pattern suggests the loss of a negative regulatory
element between positions -374 and -275. The model system for
the suppression of lymphoid-specific promoter activity in
fibroblasts is found in the IgH locus. As previously stated,
the pES motif represses the enhancer activity of the pE3
element in fibroblasts, but not in lymphoid cells (25). While
there is no consensus pES motif in the CD5 promoter, the
fibroblast transfection data suggests the loss of a pES-like

element between positions -374 and —275.

DISCUSSION

The promoters of the B-cell-specific genes mb-l and B29,
share many characteristics with the murine CD5 promoterx They
are all surface glycoproteins expressed.on B-lineage cells and
as previously stated, lack TATA boxes. All initiate
transcription from multiple sites in and around an INR-like
sequence. The promoters of all three genes contain Ig
enhancer elements that may be capable of conferring lymphoid
specificity' (20,21,31). The B29 promoter has the most
extensive collection of Ig enhancer sites, including IgH pE4,
pE3, Ig octamer/heptamer (5’—ATTTGCAT-3’) motifs and a py
tract (31). The octamer and pE elements have been found to
confer B-cell specific activity to Ig promoters and are
capable of binding proteins in a variety of cell types (23).
Deletion of the py tract upstream of octamer/heptamer sites
has been found to reduce promoter activity 2-3 fold in B-cells

(25). While the mb-l promoter contains a near-perfect octamer

73

motif, data suggests that tissue-specificity may be conferred
by a different cis-acting element (32) . The mb-l promoter
contains a functionally important PEA3/ets motif (5’-
CAGGAAGTG-3’), that has been shown to bind the transcription
fator MUF2 in PD36 and EL4 cell extracts (33). Subsequent
examination.has revealed that this motif can.bind.ets-1, ets-2
and PU.1 proteins in vitro. ets-l is expressed in B and T-
cells, PU.1 in myeloid and B-cell lineages, while ets—2 has
ubiquitous tissue distribution” 'These factors have been found
to bind.the PEA3/ets motif with.different affinities and could
possibly influence the tissue-specific expression of genes
containing this motif (34). The CD5 promoter contains
PEA3/ets sites at -174 (5’-AGGAAG-3’) and.at -232 (5’-CAGGAAG—
3’; Figure 2), and MUF2 has been shown to be present in PD36
cells (34). This could In; an. important enhancer' motif
involved in CD5 expression.

In summation, we have cloned and sequenced 1119 bp of the
5’—flanking region of the murine CD5 gene. This region can
serve as a tissue—specific promoter that mirrors the in vivo
expression of CD5 in lymphoid cells and lack expression in
fibroblasts. A deletional analysis reveals that
transcriptional regulation of CD5 is achieved through a
complex array of both positive and negative regulatory
elements. An IgH pE2 enhancer motif at -39 is a good
candidate as a critical element for lymphoid-specific
expression. The region between -374 and -275 may harbor a

pES-like element capable of repressing expression of CD5 in

74
fibroblasts. Additional CRP motifs at -198 and -234 PEA3/ets
motifs at -174 and -232, and TCF—2a motifs at -175 and -231
are also plausible elements for involvement in lymphoid-
specific expression. Further characterization of tissue-
specific factors interacting with enhancer motifs found in
this region. of the CD5 promoter, may' lead. to a Ibetter

understanding of control over tissue-specific expression.

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75
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Gorman, C., Moffat, L., and Howard,B. (1982) Mol. Cell.
Biol. 2, 1044-1051

Huang, H., Jones, N., Strominger, J., and Herzenberg, L.
(1987) PNAS 84, 204-208

Sanger, F., Nicklen, S., and Coulson, A. (1977) PNAS 74,
560-564

19.

20.

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222.

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25.

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76

Schwartz, R., Sonenshein, G., Bothwell, A., and.Gefter, M.
(1981) J. Imm. 126, 2104—2110

Sambrook, J., Fritsch, E., and Maniatis, T. (1989)
.Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY

Travis, A., Hagman, J., and Grosschedl, R. (1991).Mo1.
Cell. Biol. 11, 5756-5766

Zawel, L. and Reinberg, D. (1992) Curr. Opin. Cell Biol.
4, 488-495

Faisst, S. and.Meyer, S. (1992) NUcl. Acids. Res. 20, 3-26

Weiher, H., Konig, M., and Gross, P. (1983) Science 219,
626—631

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Hagman, J. and Grosschedl, R. (1992) PNAS 89, 8889-8893

CHAPTER 3

Regulation of Murine CD5 Expression

by Transforming Oncogenes

Timothy R. Weichert and Richard C. Schwartz

Department of Microbiology, Michigan State University

East Lansing, MI 488240-1101

77

 

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ABSTRACT
The murine CD5 surface antigen is a frequent marker on B-
lineage cell lines produced from bone marrow infected with v-
H-ras and v-src. Since CDS* B-cells cannot be detected in
adult murine bone marrow'by FACS analyis, the viral targets of
transformation are either a minor contaminating population of
CD5” B-lineage cells or the transforming oncogenes are
inducing the expression of CD5 on B-lineage cells not
previously expressing this markeru ‘We have found that v-H-ras
and v-src, but not v-myc, can induce the expression of CD5 on
two CDS‘ pre-B-cell lines established from murine bone marrow,
and.that this induction.correlates with.increased.steady-state
levels of CD5 mRNA. The existence of a signal transduction
pathway capable of modulating the expression of CD5 may alter
the concept that CD53" B-lineage cells represent a unique,

self-renewing subset of B-lymphocytes.

79
INTRODUCTION

The murine CD5 surface antigen (also referred to as Ly-1 or
Lyt-1) is a 67 KDa MW monomeric, membrane-associated
glycoprotein structurally homologous to the cysteine-rich
scavenger receptor family of polyanion-binding receptors
(Kipps, 1989; Freeman et al., 1990). CD5 is lymphoid-specific
in its expression, being found on all T-lymphocytes and on a
subset of B-cells found mainly in serousal cavities of adult
mice. In addition, FACS (fluorescence-activated cell sorter)
analysis has determined that small populations of CD5+ B-cells
reside in adult murine spleen and fetal liver, but not in
adult bone marrow or peripheral circulation (Kipps, 1989).
CD5 is physically associated with the TCR/CD3 complex on T-
cells where it is thought to be a phosphorylation target for
the protein tyrosine kinases p56”" and p59fyn during the
transduction of extracellular signals (Burgess et al., 1992;
Osman et al., 1992). No such function has been attributed to
B-cell-associated CD5, but the B-lymphocyte—specific surface
antigen CD72 (Lyb-2) has been identified as a ligand for CD5
(Van de Velde et al., 1991). The implications of CD72/CD5
binding for T-B and B-B-cell interactions have yet to be
elucidated.

CD5-expressing B-lineage cell lines are established at
high frequency'in.specialized.culture systems that support the
growth. of IB-lymphocytes in vitro. A. simple, long term
splenocyte culture system has been described in which

CD5‘sIgM“Ia*FcR"C3R+ B-cell lines are established after a 3 to

80
4-week crisis period (Braun, 1983). Also, a culture system
that allows establishment of CD5‘jpro-B-cell lines from bone
marrow of 4-week-old Balb/c mice has been described (Palacios
et al., 1987). In this system, murine bone marrow cells are
first depleted of myelocytes and mature lymphocytes, and then
passaged in WEHI—3 conditioned media known to contain IL-3.

CD5 expression is rare on pre-B-cell lines spontaneously
arising from Whitlock-Witte high density bone marrow cultures
established from bone marrow of 4-week-old Balb/c mice (R.
Schwartz, unpublished observations; Whitlock et al., 1983).
However, if bone marrow is first infected with a transforming
retrovirus expressing the v-H—ras oncogene and then cultured
in the Witte-Whitlock system, CD5*jpre-B-cell lines are the
sole outgrowth (Schwartz et al., 1986 and unpublished
observations). Interestingly, if bone marrow is coinfected
with v—H-ras and v-myc-expressing retroviruses and replated as
above, the resultant pre-B-cell lines are CDS‘ (Schwartz et
al., 1986 and unpublished observations).

Another method utilizing transforming retroviruses to
establish CD5’IB-lineage cell lines from bone marrow at high
frequency has been reported. In this system, bone marrow was
harvested from 2 to 5-week-old mice and infected with
replication defective retroviruses expressing either v-src, v-
H-ras, v-K-ras, v-fes, or v-abl. After infection, the cells
were suspended in soft agar. Individual colonies were picked
and then expanded in 'bulk' cultures on feeder layers for 6

weeks. .Analysis of these immortal lines for CD5 revealed that

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less than 50% of the individual cells, in a given cell line
established with v-abl, expressed CD5 (2 lines), 2 out of 6
lines established with v-src expressed CD5 and all lines
established with v-fes (3 lines total), v-H-ras (5 lines
total), or v-K-ras (1 line) expressed this marker (Pierce and
Aaronson, 1982; Holmes et al. , 1986) . This work was extended
by another group who found zero out of eleven cell lines,
established by Abelson murine leukemia virus (A-MuLV; v-abl)
infection of murine bone marrow, expressed CD5 (Tidmarsh et
al., 1989).

CD5” B-cells are thought to represent a unique lineage of
B-lymphocytes, ’B-l’ cells, which are distinct from the CDS‘
’B-2’ cells found in adult murine bone marrow, lymph nodes and
peripheral circulation (for review, see Kantor and Herzenberg,
1993; Herzenberg and Stall, 1989). The support for
classification of CD5+ B-cells as a unique lineage comes, in
part, from adoptive transfer experiments utilizing lymphocytes
derived from different immune compartments. It was found that
if adult murine bone marrow was used to reconstitute the
immune system of lethally-irradiated mice, CD5+ B-cells could
not be detected in the peritoneal cavities of the recipients.
However, if peritoneal lymphocytes were used as donor cells in
the reconstitution, donor CD5+ B-cells could eventually be
found to repopulate the peritoneal cavity (Hayakawa et al.,
1985) . The interpretation of this experiment was that CD5“ B-
cells could only be replenished from CD5“sIgM+ precursors

residing in the peritoneal cavity, and not CDS'sIgM’ pro/pre—B-

82

cells found in adult bone marrow. If this model is applied to
the data demonstrating high frequency of establishment of CD5+
B-lineage lines from murine bone marrow (utilizing
transforming retroviruses), one may conclude that there is a
small contaminating population of CD5+ B-lineage cells in bone
marrow that is preferentially susceptible to transformation by
certain oncogenes.

A necessary extension of the lineage model is that CD5
expression is a constitutive phenomenon and not available, as
a phenotype, to B-cells that do not have CD5+ precursors.
However, evidence has been accumulating that suggests that CD5
expression on murine B-cells may be subject to some
modulation. CD5 expression on the 702/3 murine pre-B-cell
line has been found to be down-regulated by IL-4 and up-
regulated 3-to-4-fold by lipopolysaccharide (LPS) (Jyonouchi
et al., 1990). Also, CD5 transcript levels are modulated as
the murine B-cell line BCL1-3B3 traverses the cell cycle
(Brooks et al., 1992). However, the most intriguing
experiment demonstrating CD5 modulation was offered by Ying-zi
et al. (1991). This group used FACS to sort against the
’typical’ CD5+ B-cell and collected a population of CD5'
sIgM‘IgDhighCDZBhighCD4Shigh cells (’B-2’ cells) from murine
spleen. USing an anti-Mu antibody, they then cross-linked
surface IgM on this population and noted an increase in the
percentage of CD5-expressing cells, as well as an increase in
actual cell numbers upon resorting. The authors claimed that

if this increase had been due to the expansion of a small

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population of contaminating CD5+ B-cells, a bd-modal
distribution would have been evident upon FACS analysis. In
their discussion, they hypothesized that there were two types
of activation pathways for 'B-2’-cells, one that was LPS-
inducible, resulting in secreted Ig with no increase in CD5
(standard B-cell activation assay), and the other that
proliferated and up-regulated CD5 expression upon anti-sIgM
crosslinking (but does not produce secreted Ig) . In addition,
treating the CD5-inducible splenic populations with
interleukin-6 yielded a surface phenotype identical to
peritoneal-derived.CD5*B-cells. flfimzauthors interpreted this
to mean that the CD5 antigen may be a marker for proliferation
of ’B-2' cells and that CD5" B-cells may not represent a
distinct lineage.

What model, then, is most easily reconciled with the data
demonstrating oncogene-mediated, high frequency establishment
of CD5‘IB-lineage cell lines from murine bone marrow? If the
targets of transforming retroviruses expressing v-H-ras are
restricted to CEB‘ B-lineage cells, then the lineage model
would be more compelling. However, if the retroviral targets
are more diverse and include CD5“ B—cells, then the cell line
establishment data would be most reconcilable with the idea
that the CD5 phenotype is available to any immature B-cell.
If this were the case, it may be possible that the oncogenes
expressed in these CDS‘ B-cells are activating a signal
transduction pathway that results in the induction of CD5

expression.

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While the work demonstrating CD5 up—regulation on sorted
populations of splenic 'B-2' lymphocytes (Yin—zi et al., 1991)
is very solid, one technical objection is evident. This work
relied upon the purification of ’B-2’ cells, from heterogenous
B-cell populations, that presumably included CD5+ B-cells. It
is formally possible, although unlikely, that what was
interpreted as an induction of CD5 expression, could actually
have been the expansion of a contaminating population of CD5+
B-cells. The use of clonal cell lines of the CDS‘ B-lineage
would obviate this objection.

In this paper we present evidence that the v-H-ras and v-
src oncogenes, but not the v-myc oncogene, is capable of
inducing CD5 expression in clonal pre-B-cell lines derived
from adult murine bone marrow. Also, in an attempt to define
a mechanism for this induction, we have utilized the recently
characterized murine CD5 promoter (Weichert and Schwartz,
manuscript in preparation) to elucidate signal transduction

events converging on the CD5 gene.

RESULTS
CD5-Expressing Pre-B-Cell Lines Are Established .At .High
Frequency by Infection of.MUrine Bone marrow with a v-H-ras-
Expressing Retrovirus
The establishment of four pre-B-cell lines (R1-R4) by
infection of murine bone marrow with a v-H-ras-expressing
retrovirus, and the establishment of two pre-B-cell lines (RMl

and RM2) by co-infection.with retroviruses expressing v—H-ras

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and v-myc, has been previously described (Schwartz et al.,
1986). The R1-R4 cell lines were all found to express surface
CD5 at low levels (but appropriate to CD5‘ B-cells) , while the
RMI and RM2 lines did not express CD5 (representative cell
lines R3 and RM2 shown in Figure 1). Since identical bone
marrow pools were utilized in each type of infection, the
above data might suggest that the cellular targets of
transformation are different for the v-H-ras virus, compared
to those targeted by coinfection of H-ras and with v—myc
viruses. Alternately, it is possible that the retroviral
targets are identical and that all properties including CD5
expression, among the R1—R4 cell lines, were induced by the

transforming oncogenes.

CD5 Expression in .High .Density' Bone .MarrOW' Cultures is
Conferred by the v-H-ras Oncogene

The R1-R4 cell lines were established with retroviral stocks
containing' a :replication-defective 'v-H-ras 'virus and. the
Moloney murine leukemia virus (MoMuLV) as helper. To
determine the contribution of each type of virus to
establishment of CD5‘jpre-B-cell cultures, cells were pooled
from. 10—week-old.'Witte-Whitlock. high. density' bone ‘marrow
cultures (only plates yielding >1052non-adherent cells per ml
were used), and infected with either MoMuLV alone, v-H-ras
virus containing MoMuLV’helper virus, or helper-free stocks of
v-H-ras virus. After 4 weeks in continuous culture, the

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discarded, while the other cultures flourished” IEACS analysis
of the surviving cultures demonstrated that while uninfected
cells were CD53 cultures established.with'v-H-raS'virus,*with
or without MoMuLV helper virus, expressed CD5 (Figure 2).
This demonstrates that the v—H-ras oncogene is sufficient to

confer the CD5 phenotype.

v-H-ras Can Induce CD5 Expression on a Bone Marrow-Derived
Clonal Pre-B-Cell Line

CD5 expression.on high.density bone marrow cultures may be the
result of the v-H—ras virus conferring a growth advantage to
a preferentially targeted, minor population of CEB‘ pre-B-
cells residing in murine bone marrow. Alternately, v-H-ras
may be inducing the expression of CD5 on CD5" pre-B-cells. To
determine if v-H-ras could up-regulate CD5 expression, clone
3, a CDS‘ clonal pre-B—cell line, established from murine high
density bone marrow cultures, was utilized (Whitlock et al.,
1983). Four cell populations (Clone 3R1-R4) were created by
independently infecting Clone 3 with v—H-ras virus and MoMuLV
helper virus. FACS analysis established that CD5 was faintly
expressed on the surface of the v-H-ras infected cell
populations (representative cell lines Clone 3 and Clone 3R2
shown in Figure 3). Northern analysis of cytoplasmic RNA from
Clone 3 infectants, demonstrates >5-fold induction of steady-
state levels of CD5 mRNA over the uninfected control (Figure
4). This clearly' demonstrates that v-H-ras is capable of

up-regulating CD5 expression on pre-B-cells and that this

Pi

89

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cells infected with v-H-ras

90

Figure 4 Northern analysis of CD5 expression in Clone 3 cells
infected with v-H-ras. The polyA* equivalent of 150.0 pg
cytoplasmic RNA was denatured, electrophoresed in a 1%
agarose—formaldehyde gel and transferred to a nylon membrane.
The positions of ethidium bromide-stained 288 and 183 rRNAs
are marked. The membrane was hybridized with a CD5 probe and
the position of the CD5 message (2.2 kb) is marked. The
membrane was stripped and rehybridized with a EZ-Ilglobulin
probe. Position of the Bz-pglobulin transcript (0.9 kb) is
marked. Lane 1, Clone 3, uninfected; Lane 2, Clone 3R1; Lane
3, Clone 3R2; Lane 4, Clone 3R3; Lane 5, Clone 3R4.

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FIGURE 4 Northern analysis of CD5 expression in Clone 3 cells
infected with v—H—ras

92
induction correlates with a rise in steady-state CD5 mRNA

levels.

Modulation of CD5 Expression by Transforming Oncogenes

It has been reported that infection with v-src, and possibly
the v-abl oncogene, as well as v-H-ras, can establish CD5+]B-
lineage cell lines from murine bone marrow (Holmes et al.,
1986). To examine the effect of these oncogenes on CD5
expression and to extend the observation to other CDS‘ B-
lineage cell lines, the clonal pre—B-cell line PD36
(established from adult murine bone marrow), and.the clonal B-
cell line M12 (derived from a lymphoma of an adult Balb/c
mouse), were utilized (Travis et al., 1991; Kim et al., 1979).
Initially; we repeated our examination of v-H-raS'induction.of
CD5 expression. Stably-transfected PD36 and M12 cell
populations were established by electroporation of pSV(X)-H-
ras or pSV(X)neo retroviral expression vectors, with
subsequent selection with G418. FACS analysis demonstrated
that EL4 T-cells (a positive control for CD5 staining) and
PD36ras strongly expressed.surface CD5 (Figure 5) while M12ras
showed very faint fluorescence (Figure 6). PD36 and.M12 cells
(Figures 5 and 6, respectively), as well as their pSV(X)neo-
transfected counterparts, were also very faint for CD5
staining. The above data demonstrates that up-regulation of
CD5 expression by v-H-ras is a reproducible phenomenon in at
least 2 independently-derived pre-B-cell lines, while no up-

regulation was observed in a B—cell line.

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We then extended our examination to oncogenes other than
v-H-ras that might modulate CD5 expression” Infection of PD36
cells with v-src, v-myc and H-ras helper-free retroviral
vectors was performed in duplicate. Infection with a G418-
resistant vector served as a control. Duplicate A-MuLV (v-
abl) infections of PD36 utilized MoMuLV helper virus, thus
requiring MoMuLV-infected negative controls. Northern
analysis demonstrates that the PD36 populations infected with
v-H-ras virus each expressed 14-fold more CD5 message than
uninfected PD36, and an average of 6-fold more than the
populations infected with the G418-resistant control virus
(Figure 7). PD36 populations infected with A-MuLV averaged
2.3-fold greater expression of CD5 than uninfected PD36, as
did their MoMuLV-infected counterparts. One of the PD36
populations infected.with v-src virus demonstrated a 7.7-fold
increase in CD5 expression over PD36, while CD5 transcript
levels in the other v-srtrpopulation.were not above background
(data not shown). In contrast to infected populations, the
PD36ras population, established.by electroporation of pSV(X)-
H-ras, displayed only 4.4-fold and 3-fold increases over PD36
and PD36neo, respectively (Figure 8). This suggests that
introduction of retroviral vectors by infection, rather than
electroporation, may allow more efficient expression of the
oncogenes involved in CD5 modulation. PD36 populations
infected with v—Inyc virus do not express CD5 transcript
(Figure 7). The above data demonstrate that v-H-ras (all

lines tested) and v-src (1 out of 2 lines) are capable of

96

Figure 7 Northern analysis of PD36 cells infected with v-H-
ras, v-myc, A-MULV, v-src and MOMULV. The polyAf equivalent
of 150.0 pg of cytoplasmic RNA was denatured, electrophoresed
in a 1% agarose-formaldehyde gel and transferred to a nylon
membrane. The positions of ethidium bromide-stained 288 and
188 rRNAs are marked. The membrane was hybridized to a CD5
probe and the position of the CD5 transcript (2.2 kb) is
marked. The membrane was stripped and rehybridized.with a Bf-
uglobulin.probe. The position of the fig-uglobulin transcript
(0.9 kb) is marked. Lanes: 1, PD36, uninfected; 2 and 3,
pSV(X)neol and 2; 4 and 5, MoMuLVl and 2; 6 and 7, v-H-rasl
and 2; 8, v-srcl; 9 and 10, A-MLVl and 2; 11 and 12, v-mycl
and 2; E, EL4.

97

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Figure 7 Northern analysis of PD36 cells infected with v-H—
ras, v—myc, A-MuLV, v—src and MoMuLV

98

Figure 8 Nbrthern analysis of PD36 cells stably—transfected
with pSV(X) -H-ras. The polyA” RNA equivalent of 100.0 pg
cytoplasmic RNA. was denatured, electrophoresed in a 1%
agarose-formaldehyde gel and transferred to a nylon membrane.
The positions of ethidium bromide-stained 288 and 188 rRNAs
are marked. The membrane was hybridized to a CD5 probe and
the position of the CD5 transcript (2.2 kb) is marked. The
membrane was stripped and rehybridized to a fiz-pglobulin
probe. The position of the Bz-pglobulin transcript (0.9 kb)
is marked” Lanes: E, EL4; 1, PD36, untransfected; 2, PD36neo;
3, PD36ras.

99

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Figure 8 Northern analysis of PD36 cells stably transfected
with pSV(X) —H—ras

 

100
up-regulating CD5 expression on PD36 pre-B-cells.
Interestingly, G418-resistant virus and MoMuLV-infected
negative controls demonstrated a 2.3-fold increase in CD5
expression over uninfected PD36. This could represent either
a modest effect of the retroviral vector on CD5 expression or

a by-product of the infective process.

The Effect of v-H-ras on Expression From the Murine CD5
Promoter

We have previously described the cloning of the murine CD5
promoter and have demonstrated its tissue-specific expression
(Weichert and Schwartz, manuscript in. preparation). To
determine if the v-H-ras-mediated effects on CD5 expression
occurred at the transcriptional level, the full—length CD5
promoter/CAT construct was transfected into PD36ras cells. CD5
promoter expression in PD36ras cells was not greater than
control values in PD36neo cells (Figure 10).

The inability' of stably integrated ‘v—H—ras to
transactivate the CD5 promoter in PD36 cells (Figure 9),
suggests that post-transcriptional mechanisms may govern
v-H-ras-induced up-regulation of CD5 expression. Nuclear run-
on assays were unable to detect transcriptional activity at
the CD5 locus (data not shown), suggesting that expression
from the CD5 gene may be below the limits of detection for
this assay. However, these results do not conclusively prove
that v-H-ras is incapable of activating the CD5 promoter. It

is possible that v-H-ras-responsive elements lie further

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101

Figure 9 Activity of the CD5 promoter in PD36ras cells. The
-1700 pCAT-Basic deletion construct was transiently
transfected into PD36 cells stably-expressing pSV(X)-H-ras or
pSV(X)neo. The pCAT-positive control vector transfected into
PD36neo cells yielded the lowest light unit values (1.5). All
cell extract volumes were normalized to this value for CAT
reactions. ZNumbers immediately below the figure are the ratio
of [“CJChloramphenicol converted by a given cell extract to
P‘C1chloramphenicol converted by cell extracts from the -1700
pCAT-Basic vector transfected into PD36neo cells. Figure is
representative of one replication of this experiment.

.Abbreviations: -1700, -1700 pCAT-Basic deletion construct; V,
pCAT-Basic vector; ’+’, pCAT-positive control vector.

102.

   

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Figure 9 Activity of the CD5 promoter in PD36ras cells

 

".1. 3.. 1.. - .-
ii:

103
upstream than the 1.7 kb promoter fragment we have cloned.
Furthermore, v—H—ras-induction of CD5 expression may only

occur in the context of activated chromatin.

DISCUSSION
We have demonstrated that transforming retroviruses can up-
regulate the murine CD5 surface antigen on two bone marrow-
derived pre-B-cell lines, PD36 (Travis et al., 1991) and Clone
3 (Whitlock et al., 1983). This effect is consistently seen
with the v-H-ras oncogene and can be as high as 5-to-14-fold.
The v-src oncogene showed similar induction in one infected
population, but not in another. The v-abl oncogene and
MoMuLV, itself, have a more modest 2-fold effect on CD5
expression. The contrasting CD5 expression in duplicate v—
src-expressing cell populations may' be the result of a
threshold effect involving the level of expression of the
infected oncogene. Indeed, the v—src-expressing population
that fails to express CD5 has a much lower level of v-src'mRNA
than the CD5+ population (data not shown). In.any event,
these results parallel previous findings of CD5 expression in
v-H-ras transformed pre-B-cells (Holmes et al., 1986),
inconsistent CD5 espression.in v-srtrtransformed.cells (Holmes
et al., 1986), and a lack of or weak CD5 expression in v-abl
(Holmes et al., 1986; Tidmarsh et al., 1989). Most
significantly, it is now clear that v-H-ras expression can
induce CD5 expression in pre-B-cells not previously expressing

this surface antigen.

 

104

Infection with MoMuLV by itself showed a modest 2-fold
induction of CD5 mRNA levels. This may reflect MoMuLV acting
in.a separate signalling'pathway'than‘v-H-rasy or that v—H-ras
is a more efficient activator of a common pathway. In any
case, the effect of MoMuLV’is inconsequential in comparison to
the induction mediated by v-H-ras.

The two PD36 v-myc infected lines did not show up-
regulation. of CD5 (Figure '7). If v-nwm: suppression. is
dominant over v-H—ras induction, these findings would
corroborate the data showing lack of CD5 expression on the RM1
and.RM2 cell lines established.by coinfection with v-H-raS'and
v-myc viruses (Figure 1). The question of v-myc dominance is
currently under investigation in our lab.

The fact that 'v—H—ras' consistently' up-regulated. CD5
expression on PD36 cells and v-myc consistently had no effect,
suggests that a bonafide signal transduction pathway exists in
pre-B-cells that converges on the CD5 gene. It has not been
determined whether modulation of CD5 transcript levels occurs
by increasing the rate of transcription from the CD5 locus or
at a post-transcriptional level. Data showing that the CD5
promoter/CAT'constructs were not any more active in PD36 cells
stably-expressing v—H-ras, suggests a post-transcriptional
mechanism. Alternatively, the CD5 promoter may need to be in
the context of chromatin to be trans-activated by v-H-ras.

Several precedents for H-ras oncogene activation of
cellular promoters have been detailed. The AP-l (PEAl in

mouse) motif (5’-C/GTGACTC/A-3') has been shown to bind a

105
factor that is induced.by the expression of an activated H-ras
oncogene (derived from human bladder cancer) in human
erythroleukemia cells, and. to enhance expression from. a
heterologous promoter during transient transactivation assays
utilizing H-ras constructs (Yamaguchi et al., 1989). This
site was also found to be activated by phorbol esters. A
reverse PEAl motif is found at -63 in the murine CD5 promoter
(Weichert and Schwartz, manuscript in preparation). Phorbol
esters have been shown to up-regulate CD5 expression on human
T- and B-cells but not on murine lymphocytes (Miller and
Gralow, 1984; Carrera et al., 1989; Kipps, 1989). This
suggests that PEAl sites may display some heterogeneity in
their response to phorbol esters and H—ras. This is
apparently the case with the ras-responsive element (RRE) in
the murine retrotransposon NVL3. The NVL3 LTR has been shown
to contain two PEAl sites activated by phorbol esters, only
one of which is responsive to v-H-ras (Owen et al., 1990). An
overlapping ets binding site was subsequently defined and a
ras-responsive factor, capable of activating transcription in
vitro through this motif, was shown to be induced by human EJ
H-ras (Reddy, et al., 1992). While the above work was done in
fibroblasts, members of the ets family of DNA.binding proteins
have been found in T- and B-cells (etsl) and B-/myeloid cells
(etsZ). The CD5 promoter contains two potential ets binding
sites, but these are distal to the PEAl site. The PEA-1 site

at -63 in the CD5 promoter may be a candidate for addressing

 

106
the involvement of cis-acting promoter elements in v-H-ras
modulation of CD5 expression.

Interesting correlations can be drawn between control of
expression of the LFA-l surface antigen and the murine CD5
gene. LFA-l is a lymphoid—specific surface antigen involved
in cellular adhesion (while CD5 has been shown to bind the B-
cell specific surface antigen CD72, this function has only
been postulated for CD5-expressing cells in vivo). The level
of surface LFA-l has been shown to be enhanced by EJ H-ras
expression in EBV-immortalized human B-lymphoblastoid cell
lines. This up-regulation was found to occur at the level of
increased LFA-l oz-chain mRNA levels (Endo et al., 1991).
Another group found.that c-nwm:expression.down-regulates LFA-l
expression on EBV- immortalized B-cells by an unknown mechanism
(Inghirami et al., 1990). In addition, PMA up-regulates both
LFA-l and CD5 on human B-cells. IL-4 down-regulates CD5 on
murine pre-B-cell lines while upregulating LFA—l on human
lymphoblastoid lines (Miller and Gralow, 1984; Jyonouchi et
al., 1990; Rousset et al., 1989). Further insights as to the
role of H-ras and v-myc in modulation of expression of LFA-l
may be applicable to murine CD5.

We have shown that the CD5 phenotype is available to pre-
B-cells via v-H-ras expression. This is in direct conflict
with the classification of CD5 B-cells as a unique lineage of
B-lymphocytes on a number of points. First, PD36 populations
stably-expressing v-H-ras are CD6+ and were not derived from

CD5‘sIgM“ precursors. Adoptive transfer experiments showing

 

107

the inability of murine bone marrow donor cells to regenerate
CD5+ lB-cells in lethally-irradiated mice indicates that
production of CDS*;mewB-cells by bone marrow is not possible
(Hayakawa, et al., 1985). Second, the lineage model suggests
that CD5*jpre-B-cells should not exist at all in adult mice.
Data on generation of CD5’chell lines from.murine splenocyte
cultures alone contradicts this (Braun, 1983). Thirdly, the
consistent effects on CD5 expression by v-H-ras suggest that
a signal transduction pathway capable of modulating CD5 levels
exists in pre-B-cells. This is in contrast to the lineage
model which only allows for static CD5 levels on B-cells
(Kantor and Herzenberg, 1993).

As compelling as our data may seem, one must be wary of
applying data derived from transformed cell lines to in vivo
systems. It is formally possible that the pre-B-cell
populations found.to express CD5 after infection with the v-H-
ras retrovirus, represent an as yet uncharacterized lineage
capable of CD5 modulation. However, both peritoneal-derived
CD5+ B-cells and CD5+ pre-B-cells derived from infection of
murine bone marrow with v-H-ras, have been found to have
myeloid characteristics (Kipps, 1989; Bretz et al., 1992)
suggesting that they belong to related lineages. One could
also argue that the cell lines are displaying lineage
promiscuity and that the overlapping phenotypes of CD5
expression on sIgM" B-cells are a property of the transforming
oncogenes and may never occur in vivo (Greaves et al., 1986).

While this is a valid point, it is doubtful that the existence

 

109

cultured without feeder-layers. Virus stocks consisted of
culture medium from NIH3T3 cell lines that had been
cotransfected with a proviral clone of MoMuLV and p120abl
(Rosenberg, 1982). Helper-free virus stocks consisted of
culture medium from the Psi2 cell line (Mann et al., 1983)
that had been transfected with pSV(X)—H-ras, pMMCV-neo,
(Schwartz et al., 1986), pSV(X)neo (pZipSV(X)1 of Cepko et
al., 1984) or p-src (Nori et al., 1991). Infections utilized
105 cells per ml culture media added to an equal volume of
virus stock and Polybrene (Sigma Chemical Co., St. Louis MO,
USA) to a final concentration of 8.0 pg mlfi. After a 3 hour
incubation at BTKL the cells were pelleted and resuspended in
culture medium. If infections utilized helper-free stocks,
G418 antibiotic (Gibco, Inc., Grand Island, NY, USA) was added
to a final concentration of 15.0 pg per ml for PD36 cells or
8 pg per ml for M12 cells.

Electroporation was also utilized to stably introduce
pSV(X)-Ha-ras and pSV(X)neo vectors into PD36 and M12 cells.
107 cells were suspended in 0.5 ml RPMI 1640 media
supplemented with 10% FCS (without 2ME or pen/strep) and 10.0
pg of the relevant vector. Electroporations were carried out
utilizing a 0.4 cm electrode gap cuvette in a Bio-Rad Gene
Pulser (Bio-Rad, Inc., Hercules, CA" USA) at 280 volts and 960
pF capacitance. Pulse times varied between 25 and 35 msec.
Cells were transferred to 6 cm tissue culture plates in 5 ml
complete media. At 24 hours, G418 was added to plates as

above. Successful introduction of vectors was confirmed by

 

110
Northern blot analysis of virally encoded transcripts (data
not shown).

For transiently-transfected populations, each 0.4 cm
cuvette contained 0.5 ml RPMI media/10% FCS (without 2ME or
pen/strep) with 107 cells, 4.4 pg pGL2 luciferase reporter
plasmid (Stratagene, Inc., La Jolla, CA) as an internal
control, 5.0 pg of the appropriate CAT reporter construct
(10.0 pg of CAT reporter construct for PD36 cells stably-
expressing pSV(X)-H-ras or pSV(X)neo; Weichert and Schwartz,
manuscript in preparation) and 10.0 pg of pSV(X)-H-ras or
pSV(X)neo (M12 cells only). Electroporation parameters were
280 volts and 960 pF capacitance. Pulse times varied between.
25 and 35 msec. Electroporated cells were transferred from
cuvettes to 6 cm tissue culture plates in 5.0 ml complete
mediunn After 24 hours, transfected cells were washed in ice-
cold phosphate-buffered saline (leBS) and divided into a
1/5th portion for luciferase assays and a 4/5th portion for
CAT assays. The cells analyzed for luciferase assays were
lysed in 50.0 p1 cell culture lysis buffer (Promega, Inc.,
Madison, WI) and 20.0 p1 used for luminometry in a Turner TD-
20e luminometer (Turner Designs, Sunnyvale, CA) utilizing
100.0 pl luciferin assay' buffer (Promega). Light unit
readings were collected for 15 seconds (after a delay time of
30 seconds) and the ratio of each sample value to the lowest
sample value determined (normalized luciferase ratios).
Normalized luciferase ratios were used.toldetermine the volume

of cell extract used in the CAT reaction (Gorman et al.,

111
1983) . Cell extracts for CAT assays were prepared by freeze-
thawing cells in 0.25 M Tris-HCl for 3 cycles, then heating to
65°C for 20 mins. CAT reactions were carried out utilizing
1.0 pl of 50.0 mCi/mmol [”CJChloramphenicol, 25.0 ug of n-
butyryl Coenzyme A, with cell extract and 0.25 M Tris-HCl to
a volume of 125.0 pl, and incubated at 37°C for 10 hours.
Conversion of [14C] chloramphenicol to butyrylated forms was
directly determined from TLC plates (PE SIL G/UV; Whatman,
Inc., Hillsboro, OR) on an Ambis Radioanalytic Imaging System

(Ambis, San Diego, CA).

Nucleic Acid Isolation and Analysis

CytOplasmic RNA was isolated by an SDS/urea method (Schwartz
et al., 1986) . PolyA* RNA was selected by the method of Rave
et al. (1979) . RNAs were electrophoresed through 1% agarose-
formaldehyde gels (Rave et al. , 1979) and transferred to nylon
membranes (Sambrook et al. , 1989) . Membranes were hybridized
to probes in an aqueous solution of 4xSSC, 5x Denhardt’s
solution, 10.0 pg ml‘1 denatured salmon sperm DNA, 5.0%
dextran-sulfate and 0.045M NaPi pH 6.8 at 65°C for 12 hours.
Membranes were washed to high stringency in 0.1 x SSC and 0.1%
SDS at 65°C.

Hybridization probes were prepared by random priming

(USE, Inc., Cleveland OH, USA) with the incorporation of 5'-
[a'npldATP (3000 ci mmol‘l; ICN, INC., Costa Mesa, CA. USA)-

The V-H-ras probe was a 0.46 EcoRI fragment corresponding to

. - ' us
V-H-ras-encoding sequences of Harvey murine sarcoma Vlr

 

 

112
(Ellis et al., 1980) . The v-myc probe was a 0.8 kb HindIII—
BamHI fragment corresponding to v-myc coding sequences of the
MMCVneo vector (Wagner, et al. , 1985) . The v-src probe was a
0.8 kb PvuII fragment corresponding to v-src coding sequences
(Nori et al. , 1991) . The v-abl probe consisted of the entire
pUCab12.3 vector which contains a 2.3 kb fragment of a v-abl
genomic clone construct (Rosenberg et al., 1982). The CD5
probe was a 0.44 kb EcoRV-EcoRI fragment corresponding to CD5
cDNA sequences in the MDlO vector (Huang et al., 1987) . A 0.8
kb BamHI fragment corresponding to env coding sequences of the
Friend-MuLV was utilized to detect MoMuLV expression (Silver
and Kozak, 1986) . A 1.4 kb BglII-EcoRI fragment of pSV(X)neo
was used to detect expression of this vector in control
infections (Cepko et a1. , 1984) . The 0.55 kb PstI fragment of
Bz'flglobulin was used as a probe to assess RNA loading (Parnes

and Seidman, 1982) .

Antibodies and Cell Staining

Actively dividing cell populations that displayed greater than
99% cell viability (assayed by trypan-blue dye exclusion) were
utilized for FACS analysis. 106 cells were collected from
tissue culture plates, washed in ice-cold phosphate-buffer?"d
saline (1x PBS) and incubated with anti-CD5 antibody or an
iSOtYPe-matched negative control antibody (the negative

control antibody for Clone 3 cells was the FITC-conjugated

Second antibody) and incubated 30 minutes on ice. HDBM, PD35

and M12 cells were incubated with Fc fragment (Pharmingen: San

 

 

113
Diego CA) 30 minutes on ice before the initial incubation with
anti-CD5 antibody. Cells were pelleted, washed in 1x PBS and
resuspended in R-phycoerythrin-avidin (Pharmingen) (n:
incubated with FITC-conjugated second antibody (Clone 3 cells
only) for 30 minutes on ice. Detection of surface CD5 by FACS
analysis was accomplished on an Ortho Cytofluorograph System
50 flow cytometeror (n: a Becton/Dickinson vantage II flow
cytometer. Rat anti-murine CD5 monoclonal antibodies were
biotinylated LylF (R1-R4, RM1 and RM2 staining; J. Braun,
personal communication), FITC-conjugated 53.7.3 (Clone 3
staining; Ledbetter and Herzenberg, 1979) or biotinylated.RM-
LYt-l (HDBM, PD36, M12 and EL4 staining; Pharmingen).
Isotype-matched biotinylated negative control monoclonal
antibodies utilized were L3T4 (R1-R4, RM1 and.RM2 staining; J.
Braun, personal communication) and RM-Lyt—2B (HDBM, PD36, M12
and EL4 staining; Pharmingen) . The FITC-conjugated rabbit
anti-rat IgG second.antibody utilized for Clone 3 staining was

MY 8861 (W. Esselman, personal communication).

 

 

114
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Bretz, J., Chen, S., Redenius, D., and Schwartz, R. (1992).
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Brooks, K., Yol, Y., and Pestka, J., (1992). Ann. NY Acad.
Sci., 651, 259-260.

Burgess, K., Yamamoto, M., Praud, K., and Rudd, C. (1992) 'iwflm
PNAS, 89, 9311-9315. '

Carrera, A., Cardenas, L., Tugores, A., Alonso, M., Sanchez-
Madrid, F., and de Landazuri, M. (1989). J: Biol. Chem.,
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Cepko, C., Roberts, B., and Mulligan, R. (1984). Cell, 37, ;-
1053-1062. J

Ellis, R., De Feo, D., Maryak, J., Young, H., Shih, T., Chang,
E-, Lowy, D., and Scolnick, E. (1982). J. Virol, 36, 408-
420.

Endo, K., Borer, C., and Tsujimoto, Y. (1991). Oncogene, 6.
1391-1396.

Freeman, M., Ashkenas, J., Rees, D., Kingsley, D., Copeland,
N., Jenkins, N., and Krieger, M. (1990). PNAS, 87, 88110-
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Gorman, C., Moffat, L., and Howard, B. (1982). M011. Cell.
Biol., 2, 1044-1051.

Greaves, M., Chan, L., Furley, A., Watt, 8., and Molgaard, H-
(1986). Blood, 67, 1-19.

Hayakawa, K., Hardy, R., Herzenberg, L. and Herzenberg, L-
(1985). J. Exp. med., 161, 1554-1560.

Herzenberg, L., and Stall, A. (1989). Cold Spring.Harbor Sym.
Quant. Biol., 16, 219-226.

H01mes, K., Pierce, J., Davidson, W., and Morse, H- (1986)- J-
Emp. Med., 164, 443-457.

Huang, H., Jones, N., Strominger, J., and Herzenberg, L-
(1987). PNAS, 84, 204-208.

Inghirami, G., Grignani F., Sternas, L., Lombardi, L.,
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Kantor, A., and Herzenber, L. (1993) . Annu. Rev. Immunol., 11,
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Kim, D., Kanellopoulos-Langevin, C., Merwin, R., Sachs, D.,
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SUMMARY

The ultimate goal of our work with CD5 is to more clearly
define the role of CD5” B-cells in the cellular immune system.
Toward this end, we have proceeded with an initial
investigation into the mechanisms governing murine CD5
expression in B-cells. We have taken a molecular approach,
rather than a cellular one, to address this question. The
reason for this lies in the differential expression of the CD5
gene. CD5 is expressed in a lymphoid-specific manner, being
found on all T-cells, but only on a fraction of B-cells. In

addition, it appears that expression of CD5 can be induced on

the CD5‘ B-cells. There are few, if any, surface antigens
that are expressed in this manner. Thus, by studying the
’response' (i.e. tissue-specific expression and CD5

inducibility), we will be able to more clearly define the
’stimulus’ (i.e.the immune stimuli to which CD5+ B-cells are
responding). Since all such stimuli must ultimately converge
on the CD5 locus, we have cloned and initially characterized
the CD5 promoter. We have also identified two activators of
signal transduction pathways, v-Ha-rgg and v-gg, that can up-
regulate CD5 expression.

At this point, the question to be asked is "Are CD5*IB-
cells a unique lineage of B-lymphocytes?" The answer is a
definite ’maybe’. Our data demonstrates that the CD5+
phenotype is available to pre-B-cells not previously
expressing this marker. The work of Ying-zi et al. (1991)

demonstrates that CD5 is up-regulated on B-cells activated

117

118

through their surface receptors. The above clearly shows that
CD5 is not a lineage-restricted marker. However, neither
adequately addresses the lineage status of peritoneal-derived
CD5+ B-cells. Peritoneal-derived CD5+ B-lymphocytes do not
exist in a chronic state of activation and their progenitors
are not derived from adult bone marrow (Hayakawa et al.,
1985). A way to unify our data with that of Ying-zi et al.
(1991) and Hayakawa et al. (1991), is to suggest that there
are two lineages of B-cells, one that permanently expresses
CD5 and is not bone marrow-derived, and one that can modulate
CD5 surface expression.

However, another model is formally possible. If CD5
expression is dependent on the stage of differentiation at
which a B-lineage cell received the appropriate stimulation (a
stimulus capable of inducing CD5 expression), then the
existence of a single lineage of B-cells would suffice. Our
data demonstrates that only pre-B-cells can be induced to
express CD5 by transforming oncogenes, and that B-cells are
refractive to this type of stimulation. The work of Ying-zi
et al. (1991) demonstrates that sIgM cross-linking is the type
of stimulation to which spleen-derived B—cells are responsive.
Peritoneal-derived CD5+ B-cells may have received the
appropriate stimulation at a very early stage in B-cell
differentiation and now permanently express CD5. Thus, the
ultimate factor in determination of the CD5 phenotype is
whether or not the signalling pathway that converges on the

CD5 gene, and results in CD5 up—regulation, has been

119

activated. This is the area in which our work may have the
most long-term impact. When elements that are targets for
signals that converge on the CD5 promoter have been
identified, the trans-acting' factors that activate these
elements can be identified. Identification of trans-acting
factors that are integral members of receptor-linked signal
transduction pathways will yield insights to the origin of
extra-cellular signals and, ultimately, to the immune function
of the CD5 surface antigen.
Future Directions

Two further experiments will strengthen the Northern data
that demonstrates the modulation of CD5 mRNA by transforming
oncogenes (Figure 7, Chapter 3) . The first will firmly
establish a cause-effect relationship between expression of
the v-grg oncogene and the up-regulation of CD5. PD36 cells
infected with v-srg display a 7-fold increase in steady-state
levels of CD5 mRNA (Figure 7, Chapter 3). The transforming
activity of v-grg from this retroviral construct is
temperature-dependent (Nori et al., 1991). At the permissive
temperature (37° C) , v-qu is capable of oncogenic
transformation. Changing the environment of the incubator to
the non-permissive termperature (39.5° C) will prevent
transformation by v—s_r_c. If the modulation of CD5 mRNA levels
reflects changes in v-ggg expression, a direct link between
expression of v-grg and induction of CD5 will be established.

The second experiment will address the mechanism by which

v-Ha-ras and v-myc coinfection of high density bone marrow

120
establishes cultures of (HXY' pre-B-cells, while 'v-Ha-ras
infection yields CD5+ pre-B-cells (Schwartz et al., 1986).
The above may represent v-myg suppression of v-Ha-rag-induced
CD5 up-regulation. This hypothesis can be addressed by
infecting PD36 cells that stably express v-Ha-rag, with v-myg
and assessing the level of CD5 mRNA expression. Down-
regulation of CD5 expression in the doubly-infected cells,
will solidify data suggesting that a bonafide signal
transduction pathway converges on the CD5 gene and argues
against the idea that v-Ha-rag-induced CD5 up-regulation is a

by-product of the infective process.

References

Hayakawa, K., Hardy, R., Herzenberg, L., and Herzenberg, L.
1985. Progenitors for Ly-l B cells are distinct from
progenitors for other cells. Journal of Experimental
Medicine 161: 1554.

Nori, M., Vogel, U., Gibbs, J., and Weber, M. 1991.
Inhibition of v-src-mediated transformation by'a GTPase-

activating protein. Molecular and Cellular Biology 11:
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Schwartz, R., Stanton, L., Riley, S., Marcu, K., and.Witte, O.
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Molecular and Cellular Biology 6: 3221.

Ying-zi, C., Rabin, E., and Wortis, H. 1991. Treatment of
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