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thesis entitled

Serum Responsiveness of Carbohydrate
Binding Protein 35Expression: Comparison
Between Human Fibroblasts of Different
Replicative Capacities

presented by

Kimberly K. Hamann

has been accepted towards fulfillment
of the requirements for .

 

 

Master of Sciencelegreeirl Biochemistry

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Aug. 24, 1990
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MSU Is An Affirmative ActiorVEquel Opportunity Institution
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SERUM RESPONSIVENESS OF CARBOHYDRATE BINDING
PROTEIN 35 EXPRESSION: COMPARISON BETWEEN
HUMAN FIBROBLASTS OF DIFFERENT
REPLICATIVE CAPACITIES

by

Kimberly K. Hamann

A THESIS

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

MASTER OF SCIENCE

Department of Biochemistry

1990

 

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ABSTRACT
SERUM RESPONSIVENESS OF CARBOHYDRATE BINDING
PROTEIN 35 EXPRESSION: COPMPARISON BETWEEN
HUMAN FIBROBLASTS OF DIFFERENT
REPLICATIVE CAPACITIES
BY

Kimberly K. Hamann

We have compared the expression and localization of Carbohydrate Binding
Protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When
quiescent young (passage 11) SL66 cells were treated with serum, there was a dramatic
stimulation in the expression of CBP35. This response was revealed both by an increase
in the percentage of cells positively stained with anti-CBP35 under immunofluorescence,
as well as by an increase in the amount of the protein in immunoblots. The rise in the
expression of CBP35 in proliferating cells is manifested most clearly in the nuclear
fraction, with elevation in the levels of the nonphosphorylated (pI 8.7) protein, as well as
the phosphorylated(p18.2) derivative. In contrast, older (passage 31-35) cultures of SL66
fibroblasts appear to have lost the proliferation-dependent regulation of CBP35 expression.
There was little or no down regulation of CBP35 protein upon serum deprivation of the
high passage cells. Moreover, these cells also appeared to be unresponsive to serum
stimulation, in contrast to that found in young, normal human fibroblasts. The nuclear,
unphosphorylated form of the lectin was not found in any cultures of high passage SL66

cells.

To the Lord for the strength and knowledge he has provided
To my husband, David, for his love, support and patience

And to my family for their encouragement

iii

ACKNOWLEDMENTS

I would like to express my appreciation to Dr. John Wang for all that he has
taught me. I truely respect his views as a scientist and he has been an excellent mentor
to study under.

I would like to thank all of my co-workers for their advice and support: Liz
Cowles, Jamie Laing, Neera Agrwal, Sung-Yuan Wang, Kristen Yang, Patty Voss and
Shizhe Jia.

iv

Table of Contents

Pages
List of Tables ................................................................................................. vii
List of Figures ............................................................................................... viii
Abbreviations ................................................................................................. ix
Chapter 1: Literature Review ....................................................................... 1
Introduction ........................................................................................ 2
Lectins: Definition and classifications ............................................. 3
Ctype ................................................................ 3
S-type ..................................................................................... 5
Localization ........................................................................................ 6
Extracellular ........................................................................... 8
Intracellular ............................................................................ 8
Carbohydrate Binding Protein 35 ...................................................... 9
Proliferation Dependent Expression CBP35 ..................................... 10
Protein expression .................................................................. 10
RNA expression ..................................................................... 11
Serum Response Element ...................................................... 11
In Vitro Senescence of Cultured Human Fibroblasts ...................... 12
Normal Human Cells ............................................................. 12
Werners Syndrome ................................................................ 14
References .......................................................................................... 17
Chapter 2: Expression of Carbohydrate Binding Protein 35
(CBP35) in Human Fibroblasts: Senescent
cultures Lack the Nonphosporylated Nuclear
Form. ...................................................................................... 22
Introduction ........................................................................................ 23
Experimental Procedures ................................................................... 25
Cell Culture ............................................................................ 25
Quantitation of Immunofluorescent Cells ............................. 25
Preparation of Extracts and Subcellular Fractions ............... 26
One- and Two-Dimensional Gel Electrophoresis ................. 27

Results ................................................................................................ 29
CBP 35 in Human Fibroblasts in Response to Serum
Stimulation ................................................................. 29
Expression of CBP35 in SL66 Cells of Different
Passage Number: Comparison by Immuno-
fluorescence ................................................................ 33
Expression of CBP35 in SL66 Cells of Different
' passage Number: Comparison by Immuno-
blotting ....................................................................... 37
Identification ao the Isoelectric Variants of
CBP35 in Human Fibroblasts by Two-

Dimensional Gel Electrophoresis .............................. 42

Nuclear Versus Cytoplasmic Distribution of the
Isoelectric Variants .................................................... 49
Discussion .......................................................................................... 56
References .......................................................................................... 59

vi

List of Tables

Chapter 1
1. Similar Characteristics of WS and aging .................................... p. 15
Chapter 2
1. Immunofluorescence comparison of CBP35 expression
in mouse and human fibroblasts ............................... p. 32
2. Immunofluorescence comparison of CBP35 expression
in SL66 cultures of different passages ..................... p. 36
3. Quantitative comparison of the levels of CBP35 in
SL66 cultures of different passages .......................... p. 41

4. Levels of phosphorylated and unphosphorylated

CBP35 in whole cell extracts of "young"

verses "old" SL66 cells ............................................ p. 48
5. Levels of phosphorylated and unphosphorylated

CBP35 in cytosol and nuclei of SL66

cells ........................................................................... p. 55

vii

List of Figures

Chapter 1
1. Summary of structural features of C—type
animal lectins ......................................................................... p. 4
2. Summary of structural features of S-type
animal lectins ......................................................................... p. 7

Chapter 2

1. Comparison of the immunofluorescence
staining patterns of CBP35 in serum-
starved, and serum stimulated cultures
(3'1‘3/W S/KD) ......................................................................... p. 31
2. Comparison of the immunofluorescence staining
patterns of CBP35 in serum-starved,

quiescent cultures (SL66) .......................................... p. 35
3. SDS-PAGE and immunoblotting analysis for CBP35
in extracts of human SL66 fibroblasts ...................... p. 40

4. Two-dimensional electrophoretic analysis of

CBP35 in extracts of human SL66

fibroblasts on IEF and NEPHGE .............................. p. 44
5. Comparison by NEPHGE of the isoelectric

variants in extracts of human SL66

fibroblasts ................................................................... p. 47
6. Detection of CBP35 and LDH in the cytosol

and nulei fractions of human SL66

fibroblasts ................................................................... p. 51
7. Comparison by NEPHGE of the isoelectric

variants of CBP35 in subcellur

fractions in human SL66 fibroblasts ......................... p. 54

viii

CBP35

CRD
DME
EDTA

ER

MEM

N ADH
NEPHGE
PAGE

pI

SDS

SRE

SRF

Tris

WS

Abbreviations

Carbhydrate Binding Protein 35

Chicken Lactose Lectin

Carbohydrate Recognition Domain

Dolbecco modified Eagle’s medium

Disodium Ethylenediamine Tetraacetate
Endoplasmic Reticulum

heterogeneous nuclear ribonucleoprotein complex
isoelectrice focusing

Lactate dehydrogenase

Eagle’s minimal essential medium

Nicotinamide Adenine Dinucleotide reduced form
nonequilibrium pH gradient electrophoresis
polyacrylamide gel electrophoresis

isoelectric point

sodium dodecyl sulfate

Serum Response Element

Serum Response Factor

Tris (hydroxymethyl) aminomethane

Wemers Syndrome

Xenopus Iaevis skin lectin

ix

CHAPTER 1:
LITERATURE REVIEW

INTRODUCTION

The objective of this project was to compare the expression of Carbohydrate
Binding Protein 35 (CBP; M, 35,00) in cells with different replicative capacities. In
order to address this question analysis was carried out: (a) at the level of single cells by
immunofluorescence; (b) at the level of total cellular extracts by Western blotting; and
(c) at the level of isoelectric variants by two-dimensional gel electrophoresis. The

following chapter provides the background for this project.

Lectins: Definition and Classifications

Lectins were originally isolated from plant and animal sources on the basis of their
saccharide binding (1,2,3). The present review focuses on animal lectins. The portion
of the protein involved in the sugar binding has been defined as the carbohydrate
recognition domain (CRD)(2). Some lectins also contain another domain, with properties
not necessarily related to saccharide-binding (e.g. membrane anchorage). It has been
hypothesized that many of these bifunctional lectins arose by the fusion of two separate
genes.

There are mainly two categories of animal lectins, the C-type and the S-type (2). The
C-type , or Ca” dependent, lectin is a protein which requires the presence of Ca2+ ions
for carbohydrate binding activity. In addition, the pH of the solution must be above 6.5
and the disulfide bridges must be formed between the designated cysteines ( shown in
figure 1) for the CRD to be active. Thus far, all the C-type lectins are extracellular. The
CRD is generally located on the carboxyl end of the molecule and there appears to be a
conserved motif of 18 amino acid residues that mediates carbohydrate binding. Several
of the C-type lectins are found in oligomeric structures, suggesting that they are
multivalent (3).

There are several examples of the C-type lectin. A summary is found in figure 1.
The hepatic lectins are found in a variety of species. Theyare mostly studied in the rat

and chicken. The rat hepatic lectin is also known as asialoglycoprotein receptor because

 

 

 

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Figure 1 Summary of structural features of C-type animal lectins. The (nearly) invariant
residues found in the commaon carbohydrate-meagnition domain of the C-type lectins are
shown, flanked by schematic diagrams of the special effector domains (if any) found in

individual members of the family (Referecne 2).

5
of its ability to remove desialylated glycoproteins from the circulatory system (4,5). The

rat hepatic lectin CRD has specificity for N-acetyl galactosamine where as the chicken
hepatic lectin CRD recognizes N-acetyl glucosamine (5,6). The amino terminal portion
of the polypeptide is exposed to the cytosol. The carboxyl terminal portion contains the
CRD and is exposed at the cell surface. The polypeptide thus contains a membrane
spanning domain that goes through the plasma membrane (transmembrane domain)(7).
The manose-binding protein is another C-type lectin which has specificity for manose (8).
The amino terminus is collagen like because of a triplet motif Gly-X-Y; X is often proline
and Y is frequently hydroxyproline (9,10). Yet other C-type lectins are proteoglycan
cores. The cartilage proteoglycan core has many glycosarrrinoglycan side chains attached
to its core at the amino terminal portion. It also contains a domain capable of binding
hyaluronic acids that are associated with other proteins. (11,12). The fibroblast
proteoglycan core has an amino terminus with an epidermal growth factor like-repeat; this
may suggest capability for intercellular signaling (13). Thus, the C-type lectins consist
of a wide range of proteins, with variations both in the arrrino terminal "effector" domain
as well as in the carbohydrate-binding specificity of the carboxyl terminal CRD.

The S-type (soluble or thiol dependent) lectins do not have a divalent cation
dependence in binding sugars. Rather their sulfhydral groups must be reduced in order
to bind to saccharides. These lectins contain a CRD which consists of 39 highly
conserved amino acid residues. The CRD of the S-type lectins usually have specificity
for galactoside sugars. Many of these lectins are capable of forming dimers (3).

The S-type lectin family is further subdivided into two groups: (a) carbohydrate

binding protein 13.5 (CBP13.5) and CBP16 fall into the 12-16 kDa subgroup and (b) the

6
other subgroup which consists of proteins in the 30-35 kDa range, of which CBP35 is

member. Both of these subgroups are shown in figure 2. The 12-16 kDa group contains
a single domain, the CRD. The 30-35 kDa group, on the other hand, contains a CRD in
the carboxyl terminal portion and an "effector" domain in the amino terminal portion.
CBP35 has an amino terminal domain that is glycine and proline rich and shows
some structure sinrilarities to proteins of the heterogeneous nuclear ribonucleoprotein
complex (hnRN P). The carboxyl terminal domain of CBP35 is homologous to the CRD
of other galactoside binding S-type lectins (14). Other proteins from a variety of
tissues/cell types appear to be identical or homologous to CBP35 in terms of its known
structure: (a) L-34, isolated from murine fibrosarcomas as a metastasis-associated lectin
(15); (b) Mac-2, identified as a cell surface marker for macrophage differentiation (16,17);
(c) eBP, or IgE binding protein, isolated from rat basophilic leukemia cells (18); and (d)
Laminin-Binding protein (LBP), the major non-integrin LBP from murine macrophage

(19).

Localization

The subcellular distribution of lectins may vary. Some lectins have been shown to
alter their localization depending on changes within the cell; three examples will be given
where the lectin is later found outside the cell after a cellular response. Chicken lactose
lectin I (CCL-I), a 13.5 kDa S-type lectin, has been shown to change its distribution
dependent on the stage of differentiation in embryonic chick muscle cells (25,26).

Through immunohistochemistry, CCL-I is located intracellularly in 8 day embryo

L—

 

 

 

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Figure 2: Summary of structural features of S-type animal lectins. Conserved residues
found in all of the family members so far sequenced are shown. In addition, the extra

domain found in CBP35 is shown schematically (Reference 2).

8
myoblasts but by the 12th day CCL-I is mostly extracellular upon formation of myotubes

from cell fusion. In adult chickens, CCL-I is expressed at very low levels in pectoral
muscle tissue (27). In contrast, embryonic liver tissue contains low amounts of CCL-I
and as the chick matures to an adult the expression of CCL-I is found to increase in the
lining of hepatic sinusoids (28). CLL-I’s extracellular role is suggested to be involved
in cell-cell or cell-matrix interactions by associating with proteoglycans. Chicken lactose
lectin II (CCL-II), a 16 kDa S-type lectin, also is differentially localized during
differentiation. CCL-II is found in secretory vesicles of goblet cells and is secreted with
mucin when lactose or a cholinergic stimulant is present at low concentrations (28,30,31).
Another lectin, X enopus laevis skin lectin (XL), demonstrates a change in distribution due
to an external stimulant. XL is a soluble cytoplasmic lectin (16kDa) but following an
injection of epinephrine, XL is secreted along with many other small proteins (32). These
three lectins have shown a change in distribution; this may suggest alternative roles for
the same lectin.

Many extracellular lectins have been identified and may prove to have important
roles. The Cartilage Proteoglycan core is found in the extracellular matrix of cartilage
and it is believed to be involved in forming supramolecular aggregates via the hyaluronic
acid binding site (33). The fibroblast chondoitin sulfate proreoglycan core is also
distributed in the extracellular matrix and may participate in cell-cell or cell-matrix
signaling because of the epidermal growth factor like repeat motif present in its amino
acid sequence (34).

Other localization studies have shown that lectins are distributed within the cytoplasm.

Two such lectins are calf lectin (12 kDa) and the chicken lectin (13 kDa) which were

9
found to be uniforrnily distributed in the cytoplasm (44). Previously mentioned, XL, a

Xenopus laevis skin lectin, is normally found in the cytosolic compartment (32).
Subcellular distribution of lectins in the nucleus has also been suggested. Studies
involving the use of fluorescently labeled neoglycoproteins in baby hamster kidney cells
have shown that there are specific sugar binding sites found in the nuclei. Fluorescence
analysis suggested that the sugar binding sites could be associated with RNP elements of
the nuclei (45). CBP35 is found in the nuclei and has also been found to associate with
heterogeneous nuclear ribonucleoprotein complex (hnRNP) proteins. Details of CBP35

will be covered in the following section.

Carbohydrate Binding Protein 35

CBP35 is a galactose specific lectin which was initially found in cultured 3T3
fibroblasts (50). It has since been found in a variety of mouse and human tissues,
including human SL66 skin fibroblasts (51,52).

In confluent monolayer of 3T3 cells, CBP35 has been found on the cell surface
(<5%), cytoplasm (90%) and nucleus (5%). Fluorescent studies, on the localization of
CBP35, have shown that a distinct subset of 3T3 cells exhibit punctate nuclear staining
(56). This lectin has been suggested to associate with hnRNP proteins. On cesium
sulfate gradients, CBP35 was detected at a density corresponding to that reported for
hnRNP. Nuclei from 313 cells treated with Triton X—100 release CBP35 when digested
with ribonuclease A but not deoxyribonuclease I (54). Futherrnore, the examination of

the deduced amino acid sequence of a CBP35 cDNA clone has revealed a glycine— and

10

proline-rich amino terminal portion with structural similarites to regions of hnRNP
proteins (14).

In other laboratories, some proteins have demonstrated a close if not identical
structtn'al relationship to CBP35. All these proteins have been seen on the cell surface:
L34, Mac-2, LBP, eBP (16,17,'18,19). The quantity of CBP35 seen on the surface of
3T3 fibroblasts is usually very low (<5% of total cellular CBP35). This introduces a most
puzzling question. How does CBP35 get to the cell surface since no signal sequence has
been detected for the exocytotic pathway? Several hypothesis have been presented to
explain this dual localization: (a) if this is due to secretion there must be an alternative
secretory pathway since there is no signal sequence directing it to the secretory pathway
through the ER and golgi; otherwise, (b) there must be an alternative 5’ splice site or start
site that contains the signal sequence for the exocytotic pathway, or (c) because of the
low levels in most cases, extracellular CBP35 cell distribution is due to ruptured cells.
Other proteins with a defined extracellular role have been shown to also lack the signal
sequence for secretion : (a) Basic Fibroblast Growth Factor, found in many bovine tissues
has mitogenic effect on mesodenn derived cells, (b) Factor XIII located intracellularly in
megakaryocytes and extracellularly is a blood coagulation protein, and (c) platelet derived
endothelial cell growth factor is a mitogen for endothelial cells (46,47,48). This suggests

that an alternative secretory pathway is highly probable.

Proliferation Dependent Expression of CBP35

The expression of CBP35 has been shown to be proliferation dependent in 3T3

1 1
fibroblasts. In quiescent cell, there is low level of CBP35 expressed and is mainly found

in the cytoplasm. Where in proliferating cells there is an increase in the expression of
CBP35 and it is predominantly in nucleus (53,56). Using a time course study, a
significant increase in the level of CBP35 was identified between 4 and 8 hours after
serum stimulation of serum-deprived cells (56). At the protein level, this lectin appears
to be induced early in 61 phase.

Other time course studies have confirmed the proliferation dependent expression of
CBP35 at the mRN A transcription level (55). Northern blots demonstrated a noticeable
increase in the accumulation of mRNA within 30 minutes after serum addition, followed
by a dramatic increase thirty minutes later. These data are supported by results from
nuclear run off transcription assays. Thus it appears that the gene for CBP35 is turned
on very early, shortly after the induction of c-fos (49).

Moreover, when serum stimulation was carried out in the presence or absence of
cyclohexamide, the rate of transcription and accumulation of mRN A was comparable.
In fact, an increase in the amount of detected mRNA was seen when cyclohexamide was
present (55). Such results indicate that no synthesis of other gene products is required
to stimulate transcription of CBP35. Thus, induction of CBP35 appears to be a primary
event, presumably resulting from the signal transduction of growth factors.

The sequence of a clone for the CBP35 gene has shown that this gene contains a
sequence (CCAA'I'I‘AAGG) at the 5’ flanking region that fits the consensus (CC (A/I')6
GG) termed the serum response element (SRE) (57). SRE was initially found in c-fos
and is now found in many B—actin genes. It has also been demonstrated that c-fos is

regulated by serum (58). In addition, there is an upstream site containing the SRE

12

consensus sequence in the c-fos gene (-313 to -306) that has been shown to induce
transcription upon serum stimulation (59,60). CBP35 may be regulated by an SRE and/or
it may be regulated by another means. Whichever the case, CBP35 expression has

demonstated to be proliferation dependent. This is the focus of my research.

In Vitro Senescence of Cultured Human Cells

Normal human cells have a finite replicative capacity in vitro (61,62). As cells
continue to divide they will eventually enter a stage where they no longer proliferate.
This is the state of senescence; it is irreversible (63). The in vitro senescence of cultured
human cells has been used as a model for the study of the aging process in cells, mainly
on the basis of the following correlations. First, the number of passages (population
doublings) is proportional to the life span of the donor (65). For example, a mouse has
a much shorter life span then a human being, so the proliferation capacity (number of
population doublings) of mouse cells in vitro would be significantly less than human
cultures. Second, the proliferative capacity of cultures is inversely proportional to the age
of the donor cells (64). An example of this is infant human tissues in culture have more
proliferative capacity then adult tissues in culture. Lastly, the average number of
population doubling for normal human cells is fifty (61,62).

It has been of great interest to understand what causes senescence. Some believe that
there is a molecule in senescent cells that inhibits replication by interfering with a
particular process. Others think there is a deficiency in senescent cells so that replication

doesn’t occur because of the absence of an essential molecule.

13

Evidence that anti-proliferating agents are present in senescent cells are shown in
several studies. Cell fusion studies best demonstrate this. Fusion of young proliferating
fibroblasts to senescent fibroblasts registered a decrease in DNA synthesis of the young
proliferating cells, measured by tritiated thymidine incorporation (66,67). Similar results
were seen with immortalized cells, HeLa cells (68). When poly (A)+ RNA from
senescent cells were injected into young proliferating cells, DNA synthesis was inhibited
(69). These results suggest that there is one or more proteins expressed in senescent cells
that prevent DNA synthesis.

In senescent cells, there are some studies that demonstrate an absence in certain
enzymatic activities or particular proteins. An example of a mitogenic response in
proliferating cells is the association of epidermal growth factor (EGF) associates with the
EGF—receptor (EGF-R), which results in autophosphorylation of the EGF-R in response
to EGF binding (34,36). This kinase activity is not detected in senescent WI38 cells;
however, the autophosphorylation of the platelet derived growth factor-receptor (PDGF-R)
does occur when PDGF interacts with PDGF-R (70,71). It also has been shown that
PDGF induces c-fos and c-myc expression in proliferating cells (29,58). Senescent cells
do not express c-fos when serum stimulated, but c-myc expression is similar to that of
proliferating cells (24). Normally c-fos is induced fifteen to thirty minutes after serum
addition; it is one of the first genes to be turned on after stimulation (21,22,23). The lack
of c-fos activation in senescent cells may be the reason that the cells are in a
nonreplicative state since this may eliminate a cascade of events. Several other proteins
that are expressed in proliferating cultures are not seen in senescent cultures: (a)

replication dependent histones, and (b) nuclear antigen K-67 (24,71). These proteins are

l4

expressed during S phase, so after c-fos which is expressed in 0,. Other 8 phase proteins
have been expressed in senescent cells, DNA polymerase alpha and thymidine kinase
(72,73). Many differences have been identified between replicating cultures and senescent
cultures. The cause of senescence may in part be due to the repression of c-fos which
in turn may be repressed by an anti-proliferating agent.

Premature aging cultures have also been studied. Wemers Syndrome (WS) is an
autosomal recessive human genetic disorder which is characterized by severe premature
aging signs shown in Table 1. The WS and aging don’t represent the same process;
although, they share many of the same features (78). Studies have been done in
comparing WS to normal human diploid cells. The WS patients have a more limited life
span (40). Therefore, the WS cells have a lower proliferative capacity in culture then the
normal human cells (74). Through a cell cycle study, it was revealed that the WS cells
have a cell cycle which is longer then the average growth rate for human cultures. The
extended length in the growth rate was observed to be due to a prolonged S phase. DNA
alpha polymerase was compared in WS and normal human fibroblasts. The polymerase
had the same rate of elongation in both cultures; however, the initiation rate was less in
WS cells (76).

It has been shown that WS cells undergo a very similar but accelerated senescence
compared to normal human cells (39,40). Fusion studies were done on WS cells of low
and high passage along with senescent normal human fibroblast cultures. The WS cells
were fused to normal human cells and HeLa cells. A decrease in the normal human cells

and HeLa cell DNA synthesis was seen in all fusions to WS cells, but the most significant

15
Table 1. Similar characteristics of Werner’s Syndrome and aging (R. 37)

Very similar:
1. Atherosclosis, artiosclerosis and medical calcinosis
2. Grayin of the hair
3. Hypermelanosis
4. cerebral cortical atrophy
5. Lymphoid depletion and thymic atrophy
More severe in Wemer’s Syndrome
1. Calcification of valve rings and leaflets

2. Hyalinization of serrriniferous tubes

9’

Atrophy of skin appendages

4. Osteoporosis of distal extremities

16

decreases were in cell fusions to high passage and senescent WS cells(43). These results
are similar to the fusion studies on senescent normal human cells. The morphology of
senescence appears to be the same, irregular shape and accumulated cytoplasmic fibrils
(33).

In conclusion, the significance of utilizing human tissues for studing aging has been
demonstrated by looking at senescence in normal human tissues as well as premature
aging cultures (e.g. WS). This has been shown through fusion studies, enzyme studies,

and expression studies.

10.

11.

12.

13.

14.
15.
16.
17.

18.

19.

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Monog. No. 60:241-247.

Chapter 2:
Expression of Carbohydrate Binding Protein 35
in Human Fibroblasts: Senescent Cultures

Lack the Nonphosphorylated Nuclear Form"I

22

INTRODUCTION

Carbohydrate Binding Protein 35 (CBP35; M, 35,000) is a galactose-specific lectin
found in many cells and tissues (1,2). The amino acid sequence, deduced from the
nucleotide sequence of a cDNA clone (3), showed that the polypeptide most likely
consists of two domains. The COOH-terminal half was homologous to many other [3-
galactoside—specific lectins; in particular, the conserved amino acid residues indicated that
CBP35 belonged to the S-type family of lectins (4). The NHz-terminal half was proline-
and glycine-rich and exhibited some structural similarity to proteins of the heterogeneous
nuclear ribonucleoprotein complex (hnRNP). Indeed, CBP35 has been identified in
several subcellular locations of mouse 3T3 fibroblasts, predominantly in the cytoplasm
and the nucleus (5). Analysis of the nucleoplasm by density gradient sedimentation and
by immunoblotting suggested that CBP35 is a component associated with the
ribonucleoprotein element of the cell, possibly the hnRNP complex (6).

When quiescent 3T3 cells were stimulated by the addition of serum, there was an
increased expression of CBP35 as monitored by: (a) analysis at the single cell level by
immunofluorescence (7); (b) analysis at the protein isoelectric species level by subcellular
fractionation, two-dimensional gel electrophoresis, and immunoblotting (8); (c) analysis
at the mRNA level by Northern blotting (9,10); and ((1) analysis of the transcription of
the CBP35 gene in nuclear run-off assays (10). We have recently determined the

structure of the murine CBP35 gene (11). This structure revealed a 10-nucleotide

23

24

sequence, located in the 5’ flanking region, that is consistent with the consensus sequence
for the regulatory structure designated serum response element (SRE) (12,13). Thus, this
SRE-like sequence may serve as a binding site for specific transcription factors such as
the serum response factor (SRF) and may account for the above observations on serum-
stimulated expression of CBP35.

CBP35 is also found in human fibroblasts (14,15). Because these human
fibroblasts have a finite life-span in culture, there is a well-documented, age-acquired
difference in replicative capacity between early passage (young) and late passage (old)
cells (16,17). This offered a unique opportunity to compare the expression of CBP35 in
cells with different responsiveness, in terms of DNA replication, to serum stimulation.
In preliminary studies (15), we had found that the expression of CBP35 in late passage
human SL66 fibroblasts was unresponsive to serum stimulation, in contrast to that found
in early passage normal human fibroblasts and in 3T3 cells. We now document these
observations on a quantitative basis. In addition, we also delineate one key aspect of the
difference between CBP35 in young versus old cells: the absence of the

unphosphorylated CBP35 protein in the nucleus of the old cells.

EXPERIMENTAL PRO

11 ulture

Swiss 3T3 fibroblasts (CCL92, American Type Culture Collection) were cultured
in Dulbecco modified Eagle’s medium (Gibco) containing 10% (v/v) calf serum as
described previously (10). Normal human fibroblasts, SL66 (18) and KD, were gifts from
Drs. J. J. McCormick and V. M. Maher (Michigan State University). Wemer’s Syndrome
(W S) fibroblasts AG6300 were purchased from NIA Aging Cell Repository (Camden,
NJ). The KD cells represent age-matched controls to the WS fibroblasts; the age of the
KD donor was 35 years. The human cells were all cultured in Eagle’s minimal essential
media (Gibco) containing 20% (v/v) fetal calf serum (19).

Sparse cultures of these cells were seeded at a density of 1.0 x 10‘ cells/cm2 in
their respective culture medium. The cultures were synchronized by washing twice in
media without serum and then incubated for 48 hours in their corresponding media
containing 0.2% (v/v) serum. The 3T3 cultures were stimulated by the addition of calf
serum to 10% (v/v) for 16 hours (7). The human cells were stimulated for 17 hours with

20% (v/v) fetal calf serum (15).

Quantitation 9f Immunofluorescent Cells

3T3 and human fibroblasts were seeded onto sterile coverslips as described above.
Quiescent and proliferating cultures were washed with phosphate buffered saline (.14 M
NaCl, 2.68 mM KCl, 10 rnM NazHPO.” 1.47 mM KIi,PO,, pH 7.4), and fixed onto the

coverslips with a 1 minute incubation in cold 70% acetone - 30% methanol. After

25

26
washing, the coverslips were blocked with 3% bovine serum albumin in Tris-buffered

saline (20 mM Tris, pH 7.5, 0.5 M NaCl). They were then incubated in anti-CBP35 (1:50
dilution of polyclonal rabbit antiserum) or preimmune serum (1:50 dilution) for 1 hour
at room temperature and washed, followed by incubation in fluorescein-conjugated goat
anti-rabbit immunogloban (5,7).

For quantitation of percent fluorescently-labeled cells, an arbitrary threshold level
was established to distinguish fluorescent cells from nonfluorescent cells. This threshold
level was comparable to the staining obtained with preimmune serum. For all coverslips,

several fields were counted until 100 total cells were found.

Preparation of Extracts and Sgbcellugtr Fractions

For whole cell extract preparation, the cells were washed in phosphate-buffered
saline, scraped off the culture dishes, centrifuged (1330 x g, 3 minutes) and resuspended
in 10 mM Tris (ph 7.5) containing 2 mM EDTA, 1 jig/ml soybean trypsin inhibitor, 1
Wm] aprotinin and 1 ug/rnl leupeptin. The cells were sonicated three times for 15
seconds.

For the isolation of subcellular fractions, the cells were harvested in the same way,
but resuspended in TKM buffer (20 mM Tris (pH 7.2), 5 mM KCl, 1 mM MgC11, 0.1
mM EDTA, 1 pg soybean trypsin inhibitor, 1 U/ml aprotinin, 1 ug/ml leupeptin). The
cells were lysed in a Dounce homogenizer and centrifuged (375 x g, 10 minutes). The
pellet, which contained mostly nuclei with some unbroken cells, was aspirated through
a 25-gauge needle and the resulting lysate was resedirnented to obtain the nuclear pellet

fraction. The two supematants, which contained the post-nuclear fraction, were subjected

27
to a higher speed centrifugation (150,000 x g, SW50.1, 1 hour) to yield a supernatant

(cytosolic fraction) and a precipitate (the membranous fraction) (8,20).

The total cell extracts, cytosolic fractions and nuclear fractions were measured for
protein concentration by the Bradford assay (21). The purity of the subcellular fractions
was assessed using enzyme assays for lactate dehydrogenase (LDH, EC 1.1.1.27) (22),
a cytoplasmic marker, NADH-diaphorase (EC 1.6.99.1) (23), a marker enzyme for the
endoplasmic reticulum, and the DNA binding assay for the dye, Hoechst 33258 (24). In
addition, immunoblotting analysis was also canied out using a rabbit antiserum directed
against pig muscle LDH (5); this antiserum was a gift from Dr. J. E. Wilson (Michigan

State University).

One- and Two-Qimensioni Gel Electrophoresis

Two-dimensional gel electrophoresis was carried out by using isoelectric focusing
(IEF) and nonequilibrium pH gradient (NEPHGE) electrophoresis in the first dimension,
followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE) in the second dimension (25,26). The IEF gels were prepared using 2.06
pH 3-10 ampholines (Pharmacia) and run for 10 min at 500 V and 3.5 hours at 750 V.
In NEPHGE gels, the proportion of pH 3—10 ampholines to pH 7-9 ampholines was 2:1.
The running conditions were as follows: the basic electrode solution was 50 mM NaOH;
the acidic electrode solution was 25 mM H3PO4; the running times were 60 minutes at
400 V, 75 minutes at 650 V, and 15 minutes at 750 V using a mini-gel system (BioRad).
The samples were dissolved in 9.5 M urea sample buffer and aliquots containing equal

amounts of protein (~ 100 ug) were loaded per gel.

28
SDS-PAGE analysis was performed according to the Laemmli procedure (27).

After electrophoresis, the proteins were transferred onto Irnmobilon-P (Millipore) (28).
The blots were blocked overnight in Tris-buffered saline containing 3% (v/v) bovine
serum albumin, incubated with anti-CBP35 (1:150 dilution) or with anti-LDH (1:150
dilution), washed in Tris-buffered saline containing 0.2% (v/v) Tween-80, and incubated
with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (BioRad). The
blots were quantitated using a Gelman densitometer, following methods described

previously (7,8).

RESULTS

CBP35 Expression in Human Fibroblfls in Response to Serum Stimulation

When serum-starved cultures of mouse 3T3 fibroblasts were stimulated by the
addition of serum, there was an increase in the level of CBP35, as detected by
immunofluorescence staining of the cells 16 hours after serum addition. This was
reflected both by an increase in the intensity of staining with rabbit anti-CBP35 antibodies
(Figure 1), as well as by an increase in the percentage of cells exhibiting detectable
fluorescence (Table I). Serum-starved, quiescent 3T3 cells showed little fluorescence
staining; of the stained cells (~ 35%), some showed labeling of the nucleus (Figure 1).
Sixteen hours after serum stimulation, 98% of the cells showed fluorescent staining, with
the majority (~ 85%) of the fluorescent cells stained in the nucleus. Punctate intranuclear
staining was prominent, as well as a distinct nuclear periphery.

We canied out, in parallel, the same experimental protocol on a human cell strain
designated 1(1). The staining with anti-CBP35 was weak for serum-starved KD cells
(Figure 1). Approximately half of the cells examined exhibited such weak fluorescence
(T able 1). Upon serum stimulation, the overall percentage of stained KD cells increased
to 91%. The intensity of fluorescence in the stained cells also increased (Figure 1).
Control experiments using preimmune serum were also carried out on the K1) cells.
There was negligible fluorescence due to preimmune serum in serum-starved, as well as
in serum-stimulated KD cells (Figure 1). These results indicate that the expression of
CBP35 in human KD fibroblasts was dependent on the proliferation state of the cell

culture, as was observed in mouse 3T3 cells.

29

30

Figure 1: Comparison of the immunofluorescence staining patterns of CBP35 in
serum-starved, quiescent cultures (-) and cultures 16 or 17 hours following serum
stimulation (+). ph, phase contrast; fl, fluorescence; 3T3, mouse 3T3 fibroblasts; Werner,
human fibroblasts of a Wemer’s Syndrome patient; KD, fibroblasts from a normal human
age-matched with the Werner Syndrome patient. The last column represents the staining
of human KD cells with preimmune rabbit serum in place of rabbit antiserum directed
against CBP35. Immunofluorescence was carried out on methanol-acetone fixed cells
using rabbit anti-CBP35 (1:50 dilution) or preimmune rabbit serum (1:50 dilution) and

revealed with fluorescein-conjugated goat anti-rabbit immunoglobulin.

 

31

or

5:53

 

32

Table I. Immunofluorescence comparison of CBP35 expression in mouse and

human fibroblasts"I

 

* The slides, from which representative photographs were taken for Figure l, were
subjected to counting and the numbers represent the percent of fluorescently-labeled

cells stained with anti-CBP35.

33
The behavior of human fibroblasts derived from a patient with Wemer’s Syndrome

(W S) was somewhat different from that of KD cells, in terms of the expression of CBP35
as detected by immunofluorescence. There was weak staining in about 61% of the cells
in serum-starved cultures (Figure 1 and Table 1). Addition of serum, however, failed to
increase the level of CBP35, neither in the intensity of staining in individual cells nor in
the percentage of fluorescently labeled cells. In fact, quantitation of the latter parameter
indicated that the percent of CBP35 positive cells in immunofluorescence decreased to
39% (Table I). Inasmuch as WS represents a heritable disease characterized by segmental
premature aging (29), it was of interest to compare the levels of CBP35 in a strain of
human fibroblasts that has been aged in culture, i.e. by serial passage of embryonic
fibroblasts. The normal human KD fibroblasts were used in the above study because they
represented an aged-matched control for the WS fibroblasts that were available. For the
study of CBP35 expression in fibroblasts of different passage numbers, another normal
human cell strain, SL66, was chosen because its history, in terms of number of in vitro

doublings, was known.

Expgssion of CBP35 in §_L66 Cells of Different Passage Number: Comparison by
Immunofluorescence

The staining for CBP35 was weak for serum-starved SL66 cells at low,
intermediate, and high passage numbers (passage 11, 17, and 31, respectively) (Figure 2).
Using the weak fluorescence as a criteria, we found only 8% of the young (passage 11)
cells stained with anti-CBP35. About half of the passage 17 and passage 31 cells

examined were judged to be stained (Table II).

34

Figure 2: Comparison of the immunofluorescence staining patterns of CBP35 in
serum-starved, quiescent cultures (-) and cultures 17 hours following serum stimula-
tion (+). ph, phase contrast; fl, fluorescence. Human SL66 fibroblasts at passage number
11, 17, and 31 were analyzed. The procedure described in the legend to Figure 1 was

used.

 

35

 

36

Table II. Immunofluorescence comparison of CBP35 expression in SL66 cultures

of different passages“

 

 

Passage Number Serum-Starved Serum-Stimulated
(Q) (P)
P1 1 8 98
P17 50 96
P31 55 63

 

 

 

* The slides, from which representative photographs were taken for Figure 2, were
subjected to counting and the numbers represent the percent of fluorescently-labeled

cells stained with anti-CBP35.

37

Upon serum stimulation, the percent of fluorescent cells in passage 11 cultures
increased from 8 to 98%. In individual cells, the overall staining intensity increased
dramatically, with some exhibiting punctate intranuclear staining as well as a distinct
nuclear periphery (Figure 2). Similar results were also obtained for passage 17 cultures
of SL66 cells. There was an increase in staining intensity in individual cells and the
percent labeled cells increased to 96% (Table II). These results, obtained with SL66
fibroblasts, are consistent with the corresponding observations made on normal human KD
fibroblasts and mouse 3T3 cells.

Quiescent high passage SL66 cells (passage 31) have a moderate staining intensity
and in some cases have no nuclear staining. Old SL66 cells yielded no significant
increase in immunofluorescence of CBP35 upon serum stimulation, either in terms of
staining intensity in individual cells or in terms of percent of labeled cells (Figure 2 and
Table 11). Thus, older SL66 cells (passage 31) mimicked the behavior of WS fibroblasts
in the expression of CBP35 as a function of serum addition. Overall, the present results
confirm our previous observations on CBP35 immunofluorescence in SL66 and WS
fibroblasts (15). More importantly, however, we have provided a quantitative basis for
comparison between cells of different passage numbers and between quiescent and

proliferative populations.

 

Exmssion of CBP35 in SL66 Cells of Different Passage Number: Comparison by

Immunoblotting

To compare the relative abundance of CBP35 in SL66 cells as a function of

passage number and before and after serum stimulation, extracts of SL66 cells cultured

38

under the various conditions were subjected to SDS-PAGE and immunoblotting. A single
polypeptide, migrating at a position corresponding to human CBP35 (14,15) and slightly
faster than recombinant mouse CBP35, was observed in all the extracts (Figure 3). The
intensity of the CBP35 band was quantitated by densitometric scanning and the results
are summarized in Table III. Since equal amounts of total protein (~ 100 ug) from each
extract were electrophoresed in individual lanes, the intensity of the irnmunoblotted band
reflects the amount of CBP35 per unit of total cellular protein in each sample.

In serum-starved, quiescent cultures of SL66 cells, the amount of CBP35 (per mg
of cellular protein) increased with increasing passage number of the culture. Thus, the
specific activity of CBP35 in passage 32 cells was about 9 times higher than in passage
11 cells (Table III). This is reflected, in part, by a higher percentage of anti-CBP35
positive cells under immunofluorescence in the older SL66 cells (Table II). The addition
of serum to the SL66 cultures markedly increased the amount of CBP35 in young
(passage 11) cells; this increase was approximately 9-fold on a per mg total protein basis
(Figure 3 and Table III). In the immunofluorescence analysis, both the intensity of anti-
CBP35 staining in single cells, as well as the overall percentage of stained cells increased
(Figure 2 and Table II). In intermediate passage cells (passage 17), there was a moderate
increase in the amount of CBP35 upon serum stimulation. This most probably reflects
the increase in the percent of fluorescently-labeled cells in serum-stimulated passage 17
cultures.

Finally, serum addition to passage 32 cultures of SL66 fibroblasts decreased the
amount of CBP35 (Figure 3 and Table 111). Since the percent of anti-CBP35 positive

cells under irnmunofluorescence was apparently not altered by serum stimulation of the

 

39

Figure 3: SDS-PAGE and immunoblotting analysis for CBP35 in extracts of human
SL66 fibroblasts at passage 11 (lanes 1 and 2), passage 17 (lanes 3 and 4), and
passage 32 (lanes 5 and 6). The odd-numbered lanes contained extracts derived from
serum-starved, quiescent cultures. The even-numbered lanes contained extracts derived
from cultures 17 hours after serum addition. Each lane contained 10 ug of protein.
Immunoblotting was carried out with rabbit anti-CBP35 (1 : 150 dilution) and revealed with
horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin. The position of

migration of recombinant mouse CBP35 is shown as a reference.

 

 

 

 

 

iurmn
l, and
d from
lcrivrd
urottit
xi with

ion d

 

.m>

.—

40

—
§

41

Table III. Quantitative comparison of the levels of CBP35 in SL66 cultures of

different passages“

   
   

        

1 <

 

P11 1.0 9.0
P17 ‘ 4.3 6.8
P32 l 9.2 4.3

 

* The irnmunoblots shown in Figure 3 were subjected to densitometric scanning and the

intensities of the bands are expressed as arbitrary units of CBP35.

42
culture (Table II), the observed decrease in the amount of CBP35 per unit of protein

must necessarily reflect a decrease in the CBP35 content per cell. Together, the
immunofluorescence and immunoblotting results are consistent with the notion that as
SL66 fibroblasts age in vitro, they lose the regulation of the expression of CBP35: down
regulation upon approach to quiescence due to serum starvation and up regulation upon

readdition of serum.

Idientification of the Isoelect_ric Variants of CBP35 in Human Fibroblasts by Two-
Dimensional Gel Electrophoresis

Previous studies (8) on recombinant CBP35 had identified the isoelectric point of the
mouse polypeptide to be pI 8.7 by two-dimensional gel electrophoresis. Extracts of
mouse 3T3 fibroblasts yielded two isoelectric species for CBP35, pI 8.7 and pl 8.2. We
had identified the latter to be a derivative of the pl 8.7 polypeptide due to the addition
of a single phosphate group. In these studies, two types of two-dimensional gel analysis
were used: IEF and NEPHGE (8). This was done because certain proteins with basic p1
values do not focus at the high pH end of the IEF gradient. On the other hand, the
resolution of the NEPHGE analysis at the low pH end was often poor. Moreover,
samples are usually loaded from the basic end for IEF and from the acidic end for
NEPHGE. Because of these differences, both IEF and NEPHGE analyses were carried
out in our initial search for the isoelectric variants of the CBP35 from SL66 cells.

Extracts of SL66 fibroblasts (passage 11) were subjected to electrophoresis on IEF and
NEPHGE gels, followed by immunoblotting with anti-CBP35. On IEF gels,

noimmunoreactive spots were observed (Figure 4A). Analysis of the same extract on

43

Figure 4: Two-dimensional electrophoretic analysis of CBP35 in extracts of human
SL66 fibroblasts on IEF (A) and NEPHGE (B) gels. Extracts of SL66 cells (100 pg
total protein) were electrophoresed and the protein was revealed by immunoblotting with
rabbit anti-CBP35. The numbers at the top indicate the pH values of the ampholine
gradient. The numbers at the left indicate the positions of migration of molecular weight
markers. The position of migration of recombinant mouse CBP35 is shown as a reference

for panel B.

 

 

 

 

67>

42>

rams»

‘3

 

45
NEPHGE gels revealed two spots, with pI values of 8.7 and 8.2 (Figure 4B). These

results are identical to what were observed with extracts of mouse 3T3 cells (8). On the
basis of that previous study, we infer that the pl 8.7 species represented the unmodified
CBP35 polypeptide, while the pI 8.2 species represented the phosphorylated derivative.

It should be noted that the positions of migration of the pl 8.7 and 8.2 spots remained
the same, relative to standards with known pl values (e.g. carbamylated glyceraldehyde
3-phosphate dehydrogenase, pI 8.3), irrespective of whether the NEPHGE gel was
electrophoresed for 1, 3, or 6 hours. Therefore, although the value obtained from
NEPHGE analysis may not actually represent an equilibrium isoelectric point, we will,
nevertheless, refer to the pH in the gel to which a polypeptide migrates as its pI value.

Serum-starved SL66 fibroblasts of low passage (passage 11) yielded a single isoelectric
species under NEPHGE analysis, pl 8.2 (Figure 5). Upon serum stimulation, the intensity
of this spot increased ~ 5.9-fold (Table IV). In addition, the pl 8.7 species also appeared.
Serum-starved SL66 fibroblasts of high passage (passage 35) showed a single spot (pI
8.2) on NEPHGE gels (Figure 5). The intensity of this spot was 5.5-fold greater than the
corresponding spot in serum-starved cells of low passage. Moreover, the intensity of this
pI 8.2 spot does not change appreciably upon serum stimulation in the old cells (Table
IV). These results are consistent with the previous observations that the level of CBP35
in high passage SL66 fibroblasts was rather insensitive to serum/growth state modulation.
Finally, there was no evidence of any pl 8.7 CBP35 species in serum—stimulated cultures
of passage 35 human fibroblasts (Figure 5). This is in direct contrast to the results
obtained with passage 11 human SL66 cells and with mouse 3T3 fibroblasts, in

whichextracts of serum-stimulated cells show the pI 8.7 spot.

 

46

Figure 5: Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
cultures of human SL66 fibroblasts synchronized by serum starvation (Q) and by
serum stimulation (P). P11, SL66 cells at passage 11; P35, SL66 cells at passage 35.
Approximately 100 11g of each sample were electrbphoresed and the protein was detected
by immunoblotting with rabbit anti-CBP35. The positions of migration of the pl 8.2 and

the pI 8.7 species are indicated at the top.

P11'

P35

8.7

47

04

0.7

 

48
Table IV. Levels of phosphorylated and unphosphorylated CBP35 in whole cell

extracts of "young" versus "old" SL66 cells“

 

 

 

Extract Serunzgrirv ed Scmm-Sltjrulatcd
P183 pI 87 p182 D; 3.7

P11 1.0 --_ 5.9 14

P35 5.5 --- 4.4 ---

 

 

 

 

 

* The irnmunoblots shown in Figure 5 were subjected to densitometric scanning and the

intensifies of the pI 8.2 and pI 8.7 spots are expressed as arbitrary units of CBP35.

49

Nuclear Versus Qfioplasmic Distribution of the Isoelectric Variants

The cells of passage 11 and passage 35 cultures were also subjected to subcellular
fractionation to yield a cytosol fraction (150,000 x g supernatant) and a nuclear pellet
The two subcellular fractions of passage 11 cultures were assayed by immunoblotting for
CBP35 and for LDH, a cytosolic enzyme marker. The cytosol, which accounted for .~
50% of the total protein, contained both CBP35 and LDH, as revealed by the immunoblot
(Figure 6). Approximately 25% of the cellular protein was found in the nuclear pellet;
the remainder of the protein was in membrane components that did not fractionate into
either the 150,000 x g supernatant or the nuclear pellet. The nuclei yielded a Mr ~ 35,000
band in the anti-CBP35 blot, but showed no reactive bands whatsoever in the anti-LDH
blot (Figure 6). These results, as well as marker enzyme and Hoechst dye binding assays,
indicated that there appeared to be little or no contamination of the nuclear fraction by
cytoplasmic components.

The cytosolic fraction from both serum-starved and serum-stimulated passage 11
cultures yielded only the pI 8.2 spot (Figure 7). The intensity of this spot in the cytosol
of serum-stimulated cultures was 4.7-fold higher than that of the corresponding spot from
serum-deprived cultures (Table V). This reflects the overall increase in the expression
of the CBP35 polypeptide in proliferating cells. While the nuclei of serum-starved
passage 11 cells showed only the pI 8.2 spot, the nuclei of serum-stimulated cells showed
both the pI 8.2 as well as the pl 8.7 spots (Figure 7). The intensity of the pI 8.2 spot was
about 4-fold higher in the nuclear fraction of serum-stimulated cultures than the

corresponding fraction of quiescent cultures (Table V). Thus, the increased expression

 

50

Figure 6: Detection of CBP35 and lactate dehydrogenase in the cytosol and nuclei
fractions of human SL66 (passage 11) cells. Approximately 20 ug of protein from the
cytosol (cyt) and the nuclei (nuc) fractions were subjected to SDS-PAGE in each lane.
C: Immunoblotting with rabbit anti-CBP35. L: Immunoblotting with rabbit anti-lactate
dehydrogenase. The numbers indicate the positions of migration of molecular weight

markers.

 

51

at:

‘ I
nit

I
J

2
«Wu Arte

52

of CBP35 in proliferating cells is manifested most clearly in the nuclear fraction, with
increases in both the pI 8.7 as well as in the pl 8.2 isoelectric variants.

The subcellular fractionation studies on passage 35 cells substantiate the conclusion
obtained with whole cell extracts. First, there was no increase in the level of the pI 8.2
species between serum-deprived and serum-stimulated cultures, in either the cytosolic or
the nuclear fractions (Table V). More strikingly, no pl 8.7 species was observed in the
passage 35 cells (Figure 7). This conclusion was true in examining the cytoslic or nuclear
fractions of serum-stimulated cultures, as well as the corresponding fractions of serum-

starved cells.

53

Figure 7: Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
serum-starved (Q) and serum-stimulated (P) cultures of human SL66 fibroblasts at
passage 11 (P11) and passage 35 (P35). Cyt, cytosol fraction; Nuc, nuclear pellet.
Approximately 100 pg of each sample were electrophoresed and the protein was detected
by immunoblotting with rabbit anti-CBP35. The positions of migration of the pI 8.2 and

pI 8.7 species are indicated at the top.

 

 

 

 

t
P11 a,

CyI
P35

54

55
Table V. Levels of phosphorylated and unphosphorylated CBP35 in cytosol and

nuclei of SL66 cells"

SerumStrmulated
(P)

p182 p187

cytosol 1.0 4,7 ---
nuclei 7.1 28 8. 4

M . ._ Serum-Starved ’ . , I Semm-Sumulated V_
“P35 (Q) (P)
pr 8 2 pr ‘8 7 * pl 3 2 9187

 

 

 

 

 

cytosol ' 1.4 , 1.7
nuclei f 1.6 , 1.0 ---

 

 

 

* The irnmunoblots shown in Figure 7 were subjected to densitometric scanning and the

intensities of the pI 8.2 and pl 8.7 spots are expressed as arbitrary units of CBP35.

DISCUSSION

Using a highly specific antiserum directed against CBP35, we have analyzed the
expression of this lectin at the protein level by immunofluorescence and by
immunoblotting of extracts/subcellular fractions of human fibroblasts passaged in vitro.
The following key observations have been documented: (a) The level of CBP35 in young
(passage 11) SL66 fibroblasts is low in serum-deprived, quiescent cultures but increases
dramatically (~ 9-fold) 17 hours after serum stimulation. A prominent aspect of this
increase is the appearance of the unphosphorylated form of the CBP35 polypeptide in the
nucleus of serum-stimulated cells. (b) Older (passage 31-35) SL66 fibroblasts fail to
exhibit the correlation between the level of CBP35 and the proliferation state of the
culture. The level of CBP35 remained high (no down regulation) in serum-starved
passage 32 cells and serum addition resulted in a decrease rather than the expected
increase in CBP35 expression. The unphosphorylated form of the CBP35 polypeptide
was not observed in cultures of high passage SL66 cells.

These results are particularly interesting and need to be discussed in light of two
additional, more recent observations. First, we have obtained preliminary evidence, by
Northern blotting with a cDNA probe for CBP35 (3,9), that the level of the 1.3 kb mRNA
for CBP35 was elevated upon serum stimulation of young SL66 fibroblasts, but not of the
older high passage cells. Second, Seshadri and Campisi have compared the transcriptional
expression of several genes upon serum stimulation of early passage and senescent human

fetal lung (WI-38) fibroblasts (30). Although serum induced the mRNA for c-H-ras,

56

57
c-myc, and ornithine decarboxylase normally, it failed to induce the mRN A for the c-fos

protooncogene in senescent cells.

Taken in this context, it may be important to emphasize several parallels in the
regulation of expression of the genes for CBP35 and c-fos in mouse 3T3 fibroblasts. The
mRN As for both genes are elevated early upon serum addition to quiescent cultures. An
increase in the level of the c-fos mRNA is seen within 15 minutes after stimulation
(31,32); we have observed increases in CBP35 mRN A levels 30 minutes following serum
addition (10). At least part of the increase in mRNA levels of c-fos and CBP35 is due
to an increase in the rates of transcription of the two respective genes, as revealed by
nuclear run-off assays (10,31). Moreover, the increased transcriptional expression of these
genes was observed even when the stimulation of the cells was carried out in the presence
of cycloheximide (10,33). Such observations lend support to the norion that the increased
expression of the CBP35 and c-fos genes is a direct result of signals transduced by the
binding of growth factors to their plasma membrane receptors, without the requirement
of prior synthesis of other gene products. In the nucleotide sequence of the c-fos gene,
a regulatory sequence designated SRE has been identified in the 5’ flanking region, some
300 nucleotides upstream from the site of transcription initiation (12). The SRE confers
serum inducibility by binding specific transcription factors (serum response factor). Our
recent analysis of the genorrric sequence for CBP35 has revealed a candidate for a SRE-
like‘ structure (11). The sequence in the CBP35 gene, CCAATTAAGG, differs from the
consensus SRE sequence, C-C-A-A/T-A-T-A/T-AfF-GG (12,13), at one position. An A
residue in position 5 of the 10-nucleotide consensus is replaced by a T residue in the

CBP35 gene.

 

58

Our present demonstration that senescent human fibroblasts fail to activate the
expression of CBP35 adds another item to the list of similarities between the c-fos and
CBP35 genes. Thus, these two genes are distinguished from other mitogen-stimulated
and/or cell cycle-regulated genes, such as ornithine decarboxylase, thymidine kinase,
replication dependent histone 3, c-myc, and B-actin (30,34,35). The latter is of particular
interest because the B-actin gene also contains the SRE regulatory sequence and, like c-
fos, is regulated by serum response factor (13). Although the addition of serum to
senescent WI-38 fibroblasts results in a lower level of stimulation of the actin gene than
in corresponding cells at early passage, there is, nevertheless, a clear elevation of
transcription rate, as well as accumulated mRNA (30). This suggests that senescent cells
are not deficient in serum response factor or in signals that activate it. On this basis, the
failure of CBP35 and c-fos genes to respond to serum stimulation must be ascribed to a
specific transcriptional repression mechanism.

It should be noted that the older (passage 31-35) SL66 cells not only fail to
activate the CBP35 gene upon serum stimulation of serum-deprived cultures, they also
appear to have lost the down regulation of CBP35 during serum starvation. When mouse
3T3 fibroblasts and low passage SL66 cells were deprived of serum, there is a drastic
decrease in the level of CBP35 protein, as revealed by immunofluorescence and by
immunoblotting. On the other hand, high passage SL66 fibroblasts starved of serum
retain relatively high levels of CBP35, all of which exist in the phosphorylated (pl 8.2)
form of the protein in the cytoplasm. Examination of the percent of CBP35 positive cells
in intermediate passage (passage 17) and in late passage (passage 31) cells suggest that

as the cultures age in vitro, a certain fraction of the cells depart from the normal

 

59
regulatory mechanism, including the down regulation of CBP35 protein levels. This

results in about 50% of the SL66 cells to be considered positive for CBP35 in our
immunofluorescence quantitation. Whether this portion of the cell population that has lost
the regulation of CBP35 corresponds to the same population that has lost responsiveness

to serum induction of cell proliferation remains to be determined.

 

10.

11.

12.

13.

14.

15.

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