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This is to certify that the
thesis entitled
TRANSFORMATION OF NORMAL HUMAN BREAST
EPITHELIAL CELLS BY IONIZING RADIATION AND THE
EXPRESSION OF THE CELL CYCLE REGULATING GENES

presented by

 

Chia-jen Albert Liu

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Whoratory
Science

 

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Major professor

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TRANSFORMATION OF NORMAL HUMAN BREAST
EPITHELIAL CELLS BY IONIZING RADIATION AND THE
EXPRESSION OF THE CELL CYCLE REGULATING GENES

By

Chia-jen Albert Liu

A THESIS

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

MASTER OF SCIENCE
Clinical Laboratory Science
Medical Technology Program

1997

ABSTRACT

TRANSFORMATION OF NORMAL HUMAN BREAST EPITHELIAL CELLS
BY IONIZING RADIATION AND THE EXPRESSION OF THE CELL CYCLE
REGULATING GENES

By

Chia-jen Albert Liu

Based on substantial evidence that ionizing radiation is a breast carcinogen and that
certain type of human breast epithelial cells (HBEC) is more susceptible to neoplastic
transformation, experiments were conducted on two types of normal HBEC (Type I and
Type II) with low doses of X-rays. The results show that 24 clones with extended
lifespan (E. L.) were obtained from Type 11 cells from two of six HBEC cultures whereas
no E. L. clones were isolated from Type I HBEC. These E. L. clones did not become
immortal after prolonged growth or additional X-ray irradiation. The p53 and p21 were
frequently and concomitantly elevated in these E. L. clones. These E. L. clones, however,
appear to contain wild type p53 since the cells showed radiation - induced G1 arrest.
While normal and immortal HBEC expressed only the phosphorylated form of Rb. All
except one of the E. L. clones tested, expressed both phosphorylated and
unphosphorylated forms of Rb. Unlike the other cell cultures used in this study, the cell
culture which yielded most E. L. clones was found to be deficient in p16 expression. The
expression of p16 is, therefore, suspected to be correlated with susceptibility of HBEC to
X-ray induced transformation. The results suggest a profile of cells with extended
lifespan (i.e. high Rb and p53 expression and low p16 expression), a reasonable doubt
about Type II HBEC as target cells for neoplastic transformation, and a new strategy to

transform Type I HBEC by X-rays.

TO
MY PARENTS
MY WIFE—CHIU-MEI
AND
MY DAUGHTER—AMY
FOR THEIR LOVE AND SUPPORT

iii

ACKNOWLEDGEMENTS

I would like to express my profoundly appreciation to my mentor, Dr. Chia-Cheng
Chang, for his support and guidance. Under his instruction, I entered the field of cancer
research and cell biology.

I would like to represent gratefulness to the other members of my committee, Drs. John
Gerlach, David Thorne, and James Trosko. Their helpfiil advice and guidance contributed
an important part to my research and thesis. In addition to that, Dr. Gerlach gave me
advice in choosing courses; Dr. Thorne helped me out of the class problems; Dr. Trosko
inspired the concept of cancer into me and reminded me of cultural differences.

My thanks also go to Dr. Kyung-sun Kang for his assistance regarding technical
problems in my research; Dr. Brad Upham for his suggestions in presentation. The
encouragement from faculties in clinical Laboratory Science was warm and supportive. I
deeply cherish the friendships at this Laboratory and at Michigan State University.

iv

TABLE OF CONTENTS

List of Tables ......................................................................................................... vii
List of Figures ...................................................................................................... viii
List of Abbreviations .............................................................................................. x
Introduction ................................................................................ 1
Literature review ................................................................................................. 3
Risk factors of breast cancer ............................................................... 3
Ionizing radiation and breast cancer ..................................................................... 3
Genes and breast cancer ............................................................... 4
Oncogenes ............................................................................... 4
C-erbBZ ............................................................................... 4
C-myc ................................................................................ 5
Int-2 ................................................................................. 5
Tumor suppressor genes ............................................................ 6
BRCAl .............................................................................. 6
BRCA2 ............................................................................... 6
P53 .................................................................................. 7
Cell cycle regulating genes ......................................................... 10
G1 cyclins .................................................................................................... 11
p21/W AF 1 ................................................................................................... 12
Pl6/ink4a ........................................................................... 13
Rb .................................................................................... 13

In vitro neoplastic transformation of HBEC .......................................... 15

The importance of developing an in vitro transformation model ................... 15

In vitro neoplastic transformation of HBEC ...................................... 16
Methods ................................................................................................................ 17
1 Cell culture ............................................................................. 17

2 X-ray irradiation of HBEC ......................................................... 18
3.Selection of clones with extended lifespan ....................................... 18
4.Western blot ........................................................................... 19

5 Flow cytometry ........................................................................ 23
Materials ............................................................................................................... 24
Chemicals ......................................................................................................... 24
Cell culture ....................................................................................................... 25
Medium/ grth factors/ antibodies ................................................................ 26
Results ................................................................................................................... 27

1.Induction and isolation of extended lifespan (E. L.) clones from Type II
HBEC after X-ray irradiation ......................................................... 27
2. Failure to obtained E. L. clones from type I HBEC after X-ray irradiation. 37

3. Expression of genes related to cell cycle regulation ....................................... 39
A.p53 ................................................................................... 39
B.p21 ........................................................................................................ 55
C.cyclin D1 ............................................................................ 56
D.Rb ....................................................................................... 59

Discussion ..................................................................................... 63

References .............................................................................................................. 68

vi

LISTS OF TABLES

Table 1. Induction and isolation of extended lifespan (E.L.)clones from Type II
HBEC after X-ray 1rrad1at10n 33

Table 2-1. The proliferation potential of putative E.L. clones isolated from Type
II HBEC (HMElS) after X-ray irradiation ............................................ 34

Table 2-2. The proliferation potential of putative E.L. clones isolated from Type
II HBEC (HMElS) after X-ray irradiation ......................................... 35

Table 2-3. The proliferation potential of putative E.L. clones isolated from Type
II HBEC (HMEZO) after X-ray irradiation ............................................ 36

Table 3. Results of additional X-ray irradiation of BL. clones .............................. 36

Table 4. Results of the effect of repeated X-ray irradiation of Type I HBEC on
induction of E.L. clones ............................................................................. 38

Table 5. Summary of results of cell-cycle analysis for control and X-ray
irradiated E.L.clones .................................................................................. 44

vii

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.

Figure 9.

LIST OF FIGURES

The cell cycle control in 61 phase .......................................................... 13
The morphology of normal Type II HBEC (HME20) ............................. 26
The morphology of senescent Type II HBEC (HMEIS) ........................ 26

A Colony proliferating of Type II HBEC after 6 Gy X-ray Exposure... 27

An extended lifespan colony proliferating after X-ray irradiation ........... 27
Morphology of the E. L. cells in early passage ....................................... 28
Morphology of the E. L. cells in late passage ......................................... 29
The E. L. clone after 20 Gy X-ray irradiation .......................................... 29
p53 protein expression in E.L. clones by Western blot ......................... 36

Figure 10. Expression and localization of p53 in HME2O normal and EL. cells.. 37

Figure 11. cell cycle - MCF-7 ................................................................................. 41
Figure 12. cell cycle -M158V30 ............................................................................. 42
Figure 11. cell cycle - T47D ................................................................................... 43

viii

Figure 14. cell cycle - MlSB ................................................................................. 44

Figure 15. cell cycle - M15XA25 ............................................................................ 45
Figure 16. cell cycle - MlSXBZSLl ........................................................................ 46
Figure 17. cell cycle - M15XB25L1 ZOGy .............................................................. 47
Figure 18. cell cycle - M20LB ................................................................................ 48
Figure 19. cell cycle - M20LBX1 ............................................................................ 49
Figure 20. p21 expression in E.L. clones derived from HME15 ............................ 51
Figure 21. p21 expression in E. L. clones .............................................................. 51
Figure 22. p21 expression in E.L. clones derived form HMEZO ............................. 52
Figure 23. p53 and p21 expression level in E.L. clones ......................................... 52
Figure 24. cyclin D1 expression in E.L. clones ....................................................... 53
Figure 25. Expression and localization of cyclin D1 in E.L. clones derived from 53
HMEZO
Figure 26. Rb expression and phosphorylation in E.L. clones ............................... 54
Figure 27. Rb phosphorylation in normal and EL. cells ......................................... 55

Figure 28. p16 expression and localization in E.L. clones derived from HME20.. 56
Figure 29. p16 expression in normal and EL. cells ................................................ 56

Figure 30. Expression of p16 in different normal Type II HBEC .......................... 56

ix

LISTS OF ABBREVIATIONS

HBEC ......................... Human Breast Epithelial Cell

AT .............................. Ataxia Telangiectasia

BPE ............................ Bovine Pituitary Extract

BSA ............................ Bovine Serum Albumin

cpdl ............................ cumulative population doubling level
E. L. ........................... Extended Lifespan

EGF ............................ Epidermal Growth Factor

F BS ............................ Fetal Bovine Serum

HME ........................... Human Mammary Epithelium
HPV ........................... Human PapillomaVirus

PCNA ........................ Proliferating Cell Nuclear Antigen
RPA ............................ Replication Protein A

Introduction

Although advances in breast cancer research have been made in recent years,
including the cloning of the hereditary breast cancer genes for BRCA1 and BRCA2 (Miki
et al., 1994,Wooster et al., 1995), the etiology of breast cancer is not fully understood.
For the endogenous breast cancer risk factors reported, early menarche (Bouchardy et al.,
1990), late menopause (Ewertz et al., 1988), and obesity (Gail et al., 1989), are linked to
the cumulative exposure to estrogen (Henderson et al., 1993). Many environmental
agents (e.g. organochlorine compounds such as DDE, PCBs, chlordane, and polycyclic
aromatic hydrocarbons) suspected to cause breast cancer are similarly linked by
functioning as xenoestrogens or estrogen potentiating agents (Davis et al., 1993). The
evidence for this is not yet conclusive. However, ionizing radiation is a well-established
environmental agent known to cause breast cancer. The evidence came from excess risk
of breast cancer associated with Japanese women exposed to atomic-bomb radiation in
Hiroshima and Nagasaki (Land et al., 1991). Women treated by radiation therapy for
malignant (Pollner etal., 1993) and non-malignant conditions (Hildreth et al., 1989;
Shore etal., 1977) also have increased risk. The mechanism by which X-rays induce
breast cancer is not clear. Presumably, X-rays are capable of eliminating tumor
suppressor genes through mutation. In in vitro studies, X-ray irradiation has been found
to induce tumorgenicity in an immortal tumorigenic cell line (Kang et al., in press) and to
' immortalize normal human breast epithelial cells (HBEC)(Wazer et al., 1994). Several

possible mechanisms may mediate this transformation. Altemations in the expression of

2

several cell cycle regulating genes have been demonstrated to be plausible mechanisms in
a variety of carcinogenesis models including X-ray induced (Wazer et al., 1994). The
factors of interest include, but are not limited to, p53, Rb, p21, p16 and cyclin D1.
Recently, a cell culture method has been developed in which two types of normal HBEC,
derived from reduction mammoplasty, can be grown (Kao et al., 1995). Type II HBEC is
capable of gap junctional intercellular communication (GJIC) and expresses basal
epithelial cell phenotypes. Type I HBEC is deficient in GJIC and has luminal and stem
cell characteristics. These two types of cells differ substantially in their response to
oncogenic stimulus (SV4O Large T antigen), i.e. Type I cells have the ability to establish
anchorage independent growth and easily become immortal (Kao et al., 1995). Studies
were conducted to determine if ionizing radiation is an effective initiator of neoplastic
transformation in these two types of HBEC. Additional experiments were conducted to
determine if phenotypic changes caused by X-ray is associated with alteration in

expression and function of cell cycle regulating genes.

Literature review

Risk factors for breast cancer

Breast cancer accounts for 39% of all cancers in women and is the second leading
cause of cancer death among women in the USA. Approximately 180,000 new cases and
40,000 deaths due to this disease were estimated to occur in the USA in 1992 (Boring et
al., 1992). The chance of developing breast cancer in women by the age of 85 is one in
nine (Marshall et al., 1993).

Many risk factors for breast cancer in females have been documented (Marshall et
al., 1993). The most significant ones (relative risk more than 2) include (high vs. low):
age (old vs. young), country of birth (North American, northern Europe vs. Asia, Africa),
age at first full term pregnancy (>30 vs. <20), oophorectomy (no vs. yes), body build
after menopause (obese vs. thin), family history of premenopausal bilateral breast cancer
(yes vs. no), history of fibrocystic disease (yes vs. no) and history of primary cancer in
ovary or endometrium (yes vs. no). Many environmental agents have been associated

with breast cancer by functioning as xenoestrogens (e. g. o p'-DDT, PCBs, heptachlor and

other pesticides) or as estrogenic potentiating factors (e. g. alcohol, low fiber, fat/total

calories)(Davis et al., 1993).

Ionizing radiation and breast cancer

Among the few established environmental agents that have been associated with
breast cancer, ionizing radiation is perhaps the best established exogenous carcinogen of
breast cancer. This is supported in studies with atomic—bomb survivors in Japan and in

women treated with radiation therapy (Land et al., 1991; Pollner et al., 1993; Hildreth et

4

al., 1989; Shore et al., 1977). Ionizing radiation could induce chromosomal alterations
such as deletions and mutations (Renan, 1992). X-rays, as inducer of deletion mutations,
could eliminate either tumor suppressor genes or cell cycle regulating genes. Thus, cells
may have chances to escape proliferation control and/or cellular senescence to become
immortal. Cells have at least two ways of responding to irradiation. First, cells are
arrested in G1 and induced to repair their DNA (Lane, 1992). Alternatively, cells could
be induced to apoptosis to avoid passing the defective DNA to progeny cells (Levine,

1997).

Genes and breast cancer

It has been proposed that tumors are the result of a progressive accumulation of
mutations and epigenetic alterations of oncogenes and tumor suppressor genes
(Weinberg, 1996). Several genes have been associated with the development of breast
cancers.

Oncogenes:

C-erbB2

The c-erbBZ (neu/Her-2) gene, which is located on chromosome l7q21, encodes a
185 Kda protein tyrosine kinase which, together with EGF receptor (erbBl), erbB3, and
erbB4, is a member of the Type I receptor tyrosine kinases (Carraway etal., 1994). The
ligand heregulin has been shown to bind with high and low affinity with erbB4 and erbB3
respectively and induce heterodimerization with erbB2, thereby activating both signaling
pathways (Peles et al., 1993; Carraway et al., 1994). The gene is amplified in

approximately 30% of primary human breast carcinomas (Slamon etal., 1987; Iglehart et

5

al., 1990) and other 10 % over express c-erbBZ without amplification of the gene (Kraus
et al., 1987; Slamon et al., 1989; Hollywood et al., 1993). The amplification of the gene
indicates poor prognosis and shorter time to relapse (Borg et al., 1990).

C-myc

C-myc is a nuclear phosphoprotein, which play a role in both cell proliferation and
apoptosis. The c-myc gene is located in chromosome 8q24. C-myc has a dual function:
stimulating proliferation and inducing apoptosis (Harrington et a1, 1994). Abnormalities
in the c-myc gene were reported in about 6-32% of breast cancer and most of them are
resulted from gene amplification or rearrangement (Van der Vijver and Nusse 1991). C-
myc expression was found to be induced by estrogen in estrogen responsive human breast
cancer cells (Dubik et al., 1992) and is necessary for estrogen induction of proliferation
of breast cancer (Watson et al., 1991). Data from transgenic mice confirmed the
abnormalities of c-myc are a direct cause of breast cancer (Stewart et al., 1984). Several
studies have shown that amplification of the c-myc gene to be associated with
inflammatory carcinoma, poor prognosis and post menopause disease (Van Der Vijver

and Nusse 1991; Bems et al.,1992; Escot et al.,l986).

Int-2

The int-2 gene encodes a member of the fibroblast growth factor family (Dickson et
al., 1987). The gene is amplified in up to 23 % of human breast carcinomas (Van Der
ViJver and Nusse 1991). Amplification of int-2 has been associated with local
recurrence, age more than 50 years and the presence of lymph node metastases (Van Der

ViJver and Nusse 1991). In most of the tumors with int-2 amplification, a whole cluster

6

of adjacent genes, i.e. hst, bcl-l, is co-amplified (Y oshida et al., 1988) leading to the
speculation of the presence of another gene which can confer a selective growth

advantage when amplified.

Tumor suppressor genes

Besides the three oncogenes, many tumor suppressor genes have been associated
with breast cancer. First, there are several hereditary breast cancer genes which
contribute to an estimated 5% of all breast cancer cases, and up to 36% of diagnosed
causes before age 30 (Claus et al., 1991). These genes are briefly described below.

BRCAI

This gene was mapped to chromosome l7q21 in 1990 (Hall et al., 1990) and cloned
in 1994 (Miki et al., 1994). Mutations in BRCAl alone account for about 45% of the
families with high incidence of breast cancer and 80% of families with high incidence of
both breast and ovarian cancer (Easton et al., 1993). The gene, however, appears to play
no role in sporadic, nonhereditary form of breast cancer (Futreal et al., 1994). The
function of the gene is not clear. The gene may act as a transcription activator (Monteiro
et al., 1996), or may interact with RAD 51 to participate in nuclear processes that lead to
normal chromosome recombination and genome integrity control (Scully et al., 1997)

BRCA2

This gene was localized to chromosome 13q12-13 and cloned in 1995(Wooster et al.,
1995). Germline mutations of BRCA2 predispose both men and women to breast cancer.
Studies with BRCA2 knock out mice indicate that the gene may play a role in DNA

repair and cell proliferation by interacting with the DNA-repair protein RAD51 (Sharan

7

et al., 1997). In multiple fetal and adult tissues, the spatial and temporal pattern of
BRCA2 mRNA expression is virtually indistinguishable from that of BRCA1 (Rajan et
al., 1997)

p53

The p53 gene located on chromosome 17q13 is the most commonly mutated tumor
suppressor gene in human cancer (Hollstein et al., 1991). Li-Fraumeni syndrome which
predisposes the patient to breast cancer, sarcomas and other neoplasms was found to
carry a germline p53 gene mutation (Malkin et al., 1990). As a tumor suppressor gene,
the normal function of the gene may be inactivated by several means in tumors:

1). Mutation

This is a common way by which p53 is inactivated. About half of all human

tumors have a missense mutation or deletion of the p53 gene, and 95% of these mutations
occur in the central region (Levine, 1994). There are several hot spots for point mutation,
which are mostly located in five highly conserved blocks. The codons 175, 248, 249, 273
and 282 are the most frequently mutated sites in all tumors (Levine, 1994). Different
cancers also have different hot spots, for example, the 249 codon in hepatocarcinoma and
the 175, 248, 273 codons in breast cancer (Levine, 1994).

2). Inactivation by viral /cellular oncogenes
At least 17 viral and cellular proteins have been reported to interact with the p53 protein
(Soussi, 1995). Viral oncogenes can inactivate p53 by several mechanisms. The SV40
large T antigen binds to the p53 protein and inactivates its function. Human papilloma
virus (HPV)16 E6 Protein down regulates the level of p53 protein by enhancing its

degradation through ubiquitination with the help of E6AP (Maki et al., 1996). The most

8

interesting cellular protein is MDM2 which has a p53 binding site on intron I of the gene.
P53 transactivates MDM2. Yet, MDM2 binds to p53 (both wild type and mutant) and
inhibits the transcriptional activation activity of p53 (Momand et al., 1992).

3). Localization

Cellular localization is also important for p53 to be functional. It is believed that p53
functions only in the nucleus. There is a report that one third of breast cancer with wild
type p53 have p53 localized in the cytoplasm where it is not functional (Moll et al.,
1992). Another paper has shown changes in the localization of p53 during differentiation.
P53 is in the nucleus during differentiation, whereas it appears in the cytoplasm afier
differentiation (Elizenberg et al., 1996).

While p53 may negatively regulated by MDM2, the function of the gene may be
positively activated by protein phosphorylation (Mayr et al., 1995). The p53 gene,
containing 11 exons (Benchimoll et al., 1985), is expressed in most cells. The product of
the human p53 gene is a 393 amino acid protein which is divided into three domains: the
N-terminal transactivation domain, the core DNA binding domain and the c-terrninal
domain which contains the oligomerization, nuclear localization signal, regulatory
phosphorylation sites and non-specific DNA/RNA binding activity. In normal tissue, the
wild type p53 has a short half-life from 30 minutes to 3 hours depending on cell type
(Delmolino et al., 1993). The mutant p53 is more stable and has a longer half-life than the
wild type (Delmolino et al., 1993).

As a transcription factor, p53 transactivates the expression of p21 (El-Deiry et al.,
1993), GADD45 (Carrier et al., 1994), which plays important roles in cell cycle

regulation and maintains genome integrity, and bax (Miyashita et al., 1995), which

9

induces apoptosis in addition to MDM2 which regulates p53. The major functions of p53
are briefly described in the following:

1). Regulation of cell cycle progression and maintaining genome integrity

When cells encounter a stressor such as DNA damage, hypoxia or infection by a
virus, the cell cycle may be arrested at G1 and GZ. The G1 arrest is mediated through the
p53 induction of p21 which inhibits Gl cyclins and cdk complexes, whereas GZ arrest is
mediated through p53 induction of GADD45 which inhibits cdc2/cyclin Bl (De-Toledo
et al., 1995). The cell cycle arrest allows cells to repair damage before resumption of cell
cycle progression and thus reduces gene or chromosomal mutations. Therefore, p53 is
considered as the guardian of the genome (Lane, 1992)

2). Induction of apoptosis

p53 induces apoptosis in response to DNA damage in some cells. The mechanisms
of apoptosis could either be transcriptionally dependent or independent (White, 1996). In
transcriptionally dependent apoptosis, the activity of p53 as a transcription factor is
required (White, 1996). In this case, p53 either directly induces the death signal by
activating the transcription of Bax, and/or suppresses the expression of bcl-2 (Miyashita
et al., 1995). The functions of p53 in inducing apoptosis and in arresting the cell cycle are
independent of each other (White, 1996).

3). Enhancing fidelity of DNA replication

There is some evidence suggesting that p53 may participate in DNA repair directly.
First, p53 was found to co-localize with proliferating cell nuclear antigen (PCNA), DNA

polymerase 0t, DNA ligase and replication protein A (RPA)(Wilcock et al., 1991).

Second, aside from specific binding to DNA, p53 also nonspecifically binds to single

10

stranded and double stranded DNA (Kern et al., 1991). Third, the p53 protein was
reported to have 3’ to 5’ exonuclease activity (Mummennbrauer et al., 1996) and to

enhance DNA replication fidelity of DNA polymerase (1 (Huang et al., 1997).

4). Induction of cell differentiation

Several reports have shown that transfection of p53 into many different cell lines
induced cell differentiation (Kokunai et al., 1997: Moretti et al., 1997). Although p21 was
reported to induce cell differentiation (Jiang et al., 1995), it is possible that there are
other mechanisms involved in p53 induced differentiation. The initial level of p53 may
determine if the fate of a cell is towards apoptosis or differentiation. In HL—60 cells, high
levels of p53 induce apoptosis while lower levels of p53 induce G1 arrest and cell
differentiation (Ronen et al., 1996).

Besides the three hereditary breast cancer genes described above, the ataxia-
telangiectasia (AT) gene may be also considered as a hereditary breast cancer gene, since
the carriers of the gene had greatly enhanced the risk for breast cancer (Swift et al.,

1991). The gene, ATM (11q22-23), was found to be mutated in AT patient and may
account for 7 % or more of breast cancer incidence (Easton et al., 1993).

Cell cycle regulating genes

The time interval that one cell duplicates and divides into two is defined as the cell
cycle. One cell cycle contains four phases: G1 (gap 1), S (DNA synthesis), GZ (gap2) and
M (mitosis). Those cells that remain quiescent and do not proliferate are in G0 (resting
stage). It is believed that the cell cycle is designed to regulate faithful duplication of gene
and accurate partitioning of chromosomes. The major components that control the cell

cycle are cyclin dependent kinase (cdks), cyclins and cyclin dependent kinase inhibitors

ll

(CKIs). There are at least two checkpoints for cell cycle: one is in the Gl-S transition
(Gl checkpoint), and the other is in GZ/M transition (G2 checkpoint). The purpose of
these checkpoints are believed to serve as brakes, to block cell cycle when something
goes wrong or when cells are not ready. The main controllers of the checkpoints are cdks,
which in the G1 checkpoint are cdk2, cdk4 and cdk6. The activity of cdks depends on
cyclins association and phosphorylation. The negative control of cdks is accomplished by
cdk inhibitors, which can be divided into two families, the p21 and the p16 family. Loss
of control of the transition Gl/S may cause cells to enter S phase precociously.

Gl cyclins

The D-type cyclins are synthesized in 61 phase and are induced in response to
agents that promote re-entry into the cell cycle (Motokura et al.,1991). The main fimction
of cyclin D1 is to bind to cdk4/cdk6 and activates these cdks which then phosphorylate
the Rb, resulting in the release of the transcription factor EZF which transactivates
several critical genes required for S phase entry including thymidine kinase, c-myb,
cyclin E and E2F itself (Weinberg, 1996). In cultured cells, a cDNA of cyclin D1 can
contribute to cell transformation by complementing a defective adenovirus ElA
oncogene, indicating cyclin D1 as an oncogene (Hinds et al., 1994). Cyclin D1 has been
found to be over-expressed in various human tumors, including breast carcinomas
(Lammie et al., 1991). The proximity of the over-expressed cyclin D1 to 11q13
translocation breakpoints in B cell tumor strongly suggests its identity as the putative
“bcl-l” oncogene (Rosenberg et al., 1991). Another cyclin which regulates the G1/ S
transition, cyclin E. has been shown to be over-expressed or aberrantly expressed in

breast cancers (Keyomarsi et al., 1993).

12

p21/waf1

P21 plays an important role in cell cycle regulation. It can modulate growth arrest in
response to a variety of conditions such as DNA damage (El-Deiry et al., 1994), cell
differentiation and growth factor stimulation (Datto et al., 1995; Elbendary et al., 1994).
p21 is a universal cdk inhibitor which binds to cdk/cyclin complexes and inhibits the
activity of cdks including cyclin D/cdk4, cyclin E/cdk2 and cyclin A/cdk2 (Harper et al.,
1995). The inhibition requires multiple molecules of p21 (Zhang et al., 1994). p21 is a
down stream effector of p53 and is responsible for p53 -induced G1 arrest in response to
DNA damage (Deng et al., 1995). p21 also binds to proliferating cell nuclear antigen
(PCNA a protein required for DNA replication and repair) and inhibits the replication but
not the repair function of PCNA (Li et al., 1994). Additionally, P21 has been reported to
associate with cellular senescence and differentiation. p21 was found to be elevated in
senescent cells (Noda et al., 1994), and over-expression of p21 in cells induces
senescence and differentiation (Steinman et al., 1994 ). Recently, it has been shown that
inactivation of p21 in human fibroblasts can bypass senescence and extend the lifespan
(Brown et al., 1997). On the other hand, oncogenic ras provokes premature cell
senescence with concomitant elevation in p53, p21 and p16 (Serrano et al., 1997). The
induction of p21 can result from different mechanisms (Macleod et al., 1995). In
response to DNA damage, the p53 protein is required for p21 induction. By binding to
the promoter of p21, p53 transcriptionally activates p21 expression. On the other hand,
p21 can be induced in the absence of p53 by some grth factors, chemicals or cell

differentiation (Macleod et al., 1995; Liu et al., 1996).

l3

pl6/ink4a

pl6/ink4a is a small protein product of the CDKN2/MTSI gene located on
chromosome 9p2 1. p16 binds to the cdk4 part of cdk4/cyclin D complex and inhibits the
activity of cdk4 (Serrano et al., 1993). By preventing phosphorylation of pr , p16 can
arrest cells in the G1 phase. Unlike p21, p16 is differentially expressed in various tissues.
For breast epithelial cells, p16 levels are lower than that in other tissue (Tam et al.,
1994). The level of p16 protein oscillates during cell cycle. It reaches the highest at the
peak of DNA synthesis (Tam et al., 1994). At first, CDKN2 was found frequently deleted
in many tumors (Kamb et al., 1994). Later, several reports have shown that deletion of
CDKN2 only occurred in breast cancer cell lines, suggesting that p16 deletion is only a
result from selection in cell culture (Liu et al., 1994; Quesnel et al., 1995; Musgrove et
al., 1995; Brenner et al., 1995). There is only one instance that aberrant expression of p16
in primary breast cancer (Geradts et al., 1996). Highly expression of p16 was correlated
with inactivated pr. This led to the hypothesis that p16 acts as a negative feedback loop
for pr (Serrano etal., 1993). There are several mechanisms involved in p16
inactivation: deletion, mutation, gene rearrangement and hyper-methylation (Larsen et
al., 1996). P16 is reported to be deleted in the early event of immortalization (Reznikoff
et al., 1996; Noble et al., 1996).

Rb

Rb is a tumor suppressor gene discovered in retinoblastoma. Rb plays a critical role
in cell cycle regulation. Unphosphorylated Rb binds to and sequesters the E2F family of
transcription factors to prevent transcription of critical genes that are essential for the cell

cycle. To enter S phase, Rb should be inactivated through phosphorylation by cyclin/cdk.

14

Once phosphorylated, Rb releases E2F that transactivates several critical genes required
for entry into S phase (Weinberg et al., 1995). The target genes of E2F include cdc2,
thymidine kinase, myb, dihydrofolate reductase, cyclin B and E2F itself (Weinberg et al.,
1995). Mutations, deletions and / or introduction of viral oncoproteins can inactivate Rb.
Rb mutations are detected in many tumors including small cell lung carcinoma and breast
carcinoma. Inactivation of Rb is believed to play an important role in the formation of
carcinogenesis.

In addition to the tumor suppressor genes described above, some unidentified tumor
suppressor genes on specific chromosome arms are known to be frequently lost in breast
cancer (loss of hetrerozygosity). These include chromosome 1p, lq, 3p, 11p, l6q and 18q

(Callahan et al., 1990; Sato et al., 1990)

 

 

~~+ activate ~ inhibit

Figure 1. The cell cycle control in G1 phase

15

In vitro neoplastic transformation of human breast epithelial cells (HBEC)

The importance of developing an in vitro transformation model

Carcinogenesis is a complex multi-step (Vogelstein et al., 1988), multi-mechanism
(Weinstein et al., 1984), and multi-pathway (Callahan et al., 1990) process involving
genetic and possibly epigenetic alterations of specific sets of oncogenes and tumor
suppressors (Knudson et al., 1993). Furthermore, tumors are believed to be derived from
stem cells or early precursor cells and have a phenotype similar to normal
undifferentiated cells at that stage (stem cell theory of cancer and oncogeny as blocked or
partially blocked ontogeny theory)( Potter,197 8; Trosko and Chang 1989). The
mechanisms of carcinogenesis may be understood by comparative study of normal cells
and tumor cells or preneoplastic cells in vivo as shown by the colorectal cancer model
(Vogelstein et al., 1988). The duplication of this model for breast cancer, however, has
been hampered by the relative lack of human tissue available for study, since the tiny
samples of atypical hyperplasia or in situ carcinoma have of necessity gone to the
surgical pathologist for diagnosis (Bartow et al., 1993). On the other hand, there are
advantages for developing the in vitro transformation model. By stepwise transformation
of normal cells to tumor cells, the genetic alterations and their resulting phenotypes at
different stages of neoplastic transformation can be revealed. Furthermore, by using
different types of normal cells for transformation, the target cells for neoplastic

transformation may be identified.

16

In vitro neoplastic transformation of HBEC
Immortalization is generally preceded by extended lifespan as shown by viral or chemical
transformation of HBEC (Stampfer et al., 1988; Kao etal., 1995). It is commonly
accepted that immortalization is a critical step for tumori genesis. Several methods have
been used to immortalize HBEC in vitro: chemical, viral transfection, oncogene
transfection and irradiation. Among these, viral transfection is the most efficient. The
commonly used viruses are Simian virus 40 (SV40) and human papillomavirus (HPV).
Generally, the mechanism by which viruses immortalize cells is inactivating p53 and /or
pr by expression of viral proteins. With SV40, the large T antigen can bind to and
inactive p53 and pr; with HPV16 or HPV18, E6 and E7 proteins bind to p53 and pr
respectively. These observations suggest that inactivation of p53 and pr are critical in
immortalization. Inactivation of HBEC by these viral agents has been reported for milk
cells (Bartek et al., 1990, 1991; Garcia et al., 1991) and for organoids or cells derived
from reduction mammoplasty (Berthon et al., 1992; Van Der Haegen etal., 1992; Shay et
al., 1993). There are fewer reports on successfully immortalized human cell by ionizing
irradiation (Fushimi etal., 1997; Tsutsui et al., 1997; Wazer et al., 1994), and only one
(Wazer et al., 1994) was able to immortalize human breast epithelial cells by X-ray

irradiation.

17

Methods

The objectives of this study are 1). To determine if X-rays are effective in inducing
the initiation of neoplastic transformation of a specific type of normal HBEC, i.e. lifespan
extension and immortalization, and 2). to determine if cell cycle regulating genes are
frequently altered in these initiated cells. The specific procedures and methods are
described as follows.

1. Cell culture

First passage normal HBEC, derived from reduction mammoplasty, were thawed at
37 0 C from liquid nitrogen storage. To remove the freezing solution, the cells were
transferred to a 15 ml centrifuge tube, mixed with 5 ml MSU-l medium and pelleted at
1000 rpm for 8 minutes. The pelleted cells were added to 8 ml of MSU-l medium
supplemented with 5% FBS (Type I medium), dispersed by pipetting several times and
then transferred to a 100 mm dish (plate A). After incubation (37 OC 5% C02 and
humidified air) for 2 hours to allow the residual fibroblasts to attach on the plate, the
unattached cells in medium were transferred and centrifuged. The pelleted cells were
suspended in 8 ml MSU-l medium with 4% bovine pituitary extract (Type II medium)
and inoculated in a new 100 mm dish (plate B). The plate B was incubated over night for
Type II HBEC to attach. The next day, the remaining unattached cells in medium were
transferred and pelleted by centrifugation and then suspended in Type I MSU-l medium
and plated in a third dish (plate C). As reported previously (Kao et al., 1995), Type I
HBEC developed in Type I medium in plate C whereas Type II HBEC developed in Type
11 medium in plate B. Subculture of HBEC was accomplished by using a phosphate

buffered saline (PBS) with 0.01 % trypsin and 0.01% EDTA. The trypsin is inactivated

18

by 5% F BS after trypsinization.

2. X-ray irradiation of HBEC

The first passage Type II HBEC developed in a plate (plate B) for 3—4 days were
subcultured into three 100 mm dishes. After incubation for 4-5 days, each plate contained
about 1-2x10 6 cells (50-60% confluence). These cells were irradiated by low dose X-ray
at a dose rate of 2 Gy/m for 1 minute using a Torrex 150 Kv cabinet X-ray machine
operated at 150 Kv and 5 mA. After irradiation, the medium was changed immediately
and the cells returned to an incubator. Three days after the irradiation, the cells were
irradiated again. After repeating the treatment for three times, the cells were subcultured
with a split ratio of 1:3 to allow room for the cells to proliferate. These plates after
incubation for 7 days were exposed to the same X-ray treatment for additional 2-3 times.

3. Selection of clones with extended lifespan

After the last X-ray irradiation, the cells were incubated, and subcultured when
necessary, for colony development. The colonies were allowed to grow to 3-10 mm in
diameter. Large actively proliferating colonies were marked on dishes. Cells were rinsed
with PBS once. Glass cylinders were tapped on cello-seal grease, then carefully and

firmly put on the colonies. Trypsin solution (50-100 pl) was added to each cylinder and

warmed on a 37 °C plate for 8 minutes. The cells were pipetted several time and removed
from the cylinders by Pasteur pipettes .The isolated cells were plated in T25 flasks filled
with 10 ml Type 11 medium. Once approaching confluence, the cells were subcultured to
T75 flasks. After becoming confluent, these cells were trypsinized and counted by a
hemacytometer. 2x105 cells were inoculated into a new flask for continuous culture and

cell counting, the remaining cells were frozen for storage in liquid nitrogen.

19

The cells were propagated to determine the cumulative population doubling level
(cpdl) that each cell line can achieve. The process of one cell dividing into two cells is
defined as one cpdl. A normal human breast epithelial cell can be propagated to no more
than four million cells, i.e. 22 cpdl. We define a cell clone with extended lifespan when a
clone has achieved more than 24 cpdl. That means one cell proliferated to at least sixteen
million cells. For an immortalized cell line, the cpdl should be more than 100.

The cpdl is calculated as:

cpdl = ln(final cell number/initial cell number)/ln2

4. Western blot analysis

a. Protein extraction

1). Total protein

Cells were cultured on 100 mm dishes until confluence and lysed with 0.5 ml 20%
SDS plus 1 nM PMSF. Cells were scraped off the dishes and transferred into micro tubes
on ice. The protein lysis were sonicated 30 seconds to break genomic DNA, then

dispensed into 100 pl aliquots for storage at -20°C.

2). Separation of nuclear and cytoplasmic fractions

Cells were harvested in 3 ml PBS by a rubber policeman, transferred into a
centrifuge tube and pelleted at 1000 rpm at 4 0C for 5 minutes. After decanting the PBS,
the pelleted cells were washed with 2 ml Buffer I (10 mM HEPES, 10 mM KCl, 1.5 mM
MgC12, 0.5 mM DTT, 0.5 mM PMSF, 0.1 mM EGTA and 0.3M sucrose, pH7.9),
resuspended in 1 ml Buffer I and then transferred into Dounce A . The cells were
dounced, transferred to a microcentrifuge tube and centrifuged at 10,000 rpm at 40C for

30 minutes to separate the cytoplasms and nuclei (Dignam et al., 1983). The supernatant

20

(cytoplasm fraction) was transferred to a new tube. Both fractions were lysed with 20%
SDS plus 1 nM PMSF for western blot analysis.
b. Lowry protein assay (Bio—Rad DC protein Assay)

Six protein standards (BSA) from 0 mg/ml to 1 mg/ml were prepared in 20 % SDS

solution. The samples were five time diluted with 20 % SDS. 10 pl of samples /standards
were added to each microtube, followed by 50 p1 of solution A and 400 pl of solution B.

After mixing and reacting for 15 minutes at room temperature, the absorbance is
measured at 750 nm using a Beckman DU7400 spectrophotometer.

c. SDS -Polyacrylamide Gel Electrophoresis of Protein

A discontinuous gel system was used in this study (Molecular Cloning, Maniatis).
The apparatus used was a Bio-Rad mini gel system. The glass plates were assembled
according to the instructions of the manufacturer. Ten ml of the resolving gel solution
was prepared containing the desired concentration of acrylamide and other components
(for 10% gel, 4 ml H20, 3.3 ml 30% acrylamide mix, 2.5 ml 1.5 M Tris pH 8.8, 100 pl
10% SDS, 4 pl TEMED and 100 pl 10% ammonium persulfate). These ingredients were
added into a disposable plastic tube, mixed by vortex and poured into the gap between
the glass immediately. A layer of double distilled H20 (0.5ml) was added carefiilly to
prevent air from diffusing into gel. The gel was placed at room temperature for 30
minutes to polymerize. Afler polymerization, the gel was washed with double distilled
H20 two or three times. The 5 % stacking gel (4 ml for two) was prepared in a plastic

tube as follow: 2.7 ml double distilled H20, 660 pl 30 % acrylamide, 0.5 ml 1M Tris

pH6.8, 40 pl 10% SDS, 4 pl TEMED and 40 pl 10 % ammonium persulfate. The solution

21

was poured into the gap. The combs were inserted immediately and carefully to avoid air
bubbles. After gel polymerization, the combs were removed and the wells were washed
with ddH20 several times. The gels were mounted in electrophoresis apparatus. Tris-

glycine electrophoresis buffer [25 mM Tris, 250 mM glycine and 0.1% SDS] was filled

into reservoirs. Samples (30 pg / 10 pl) were added to an equal volume of 2x loading
buffer [4% SDS, 20% glycerol, 0.2% bromophenol blue and 2% B-mercaptoethanol],
then mixed and loaded to gels (10pl each). Gels were run at 158 V for 40 - 90 minutes.

d. Protein transfer

Whatman 3MM paper and PVDF membrane were cut (wearing gloves) into sheets
of 75mm x50mm (exact size of gels). The PVDF membrane was wetted with methanol,
rinsed with distilled H20, then soaked in transfer buffer (39 mM glycine, 48 mM Tris
base, 0.037% SDS and 20% methanol). After electrophoresis, the gels were removed
from glass plates and directly soaked in transfer buffer for 10 minutes. Blotting was done
using a BIO RAD mini trans -blot transfer cell. Gel holder cassettes were laid on a tray.
0ne wet fiber pad was placed on the cathode (black) side, covered with 3 sheets of 3 MM
papers. A gel was put on the paper carefully. The PVDF membrane was placed on top of
the gel while the gel covered with buffer. Three sheets of 3 MM paper were put on the
membrane. The paper was rolled over several time to squeeze out the air bubbles using a
pipette. The cassette was assembled after putting on another fiber pad and inserted into
electrode unit. The tank was filled with transfer buffer and inserted into a cooling ice

basket. The transfer was run at 23 volts at room temperature for overnight.

22

e. Staining the membranes
After transfer, PVDF membranes were removed from cassettes, rinsed with water,

and then soaked in Ponceaus stain for 5 minutes. The membranes were placed between
clear polypropylene for scanning into computer to confirm that equal amounts of total
protein were loaded.

f. Immunodetection

The membranes were incubated with 10 ml blocking solution (5% non-fat milk, 0.1
% Tween 20 in PBS) on a rocker at room temperature (RT) for one hr. After blocking,
the membranes were washed three times with PBS containing 0.1% Tween 20 (PBST) for
10 minutes on a rocker. The membranes were incubated with 5 ml primary antibodies,
diluted 1:1000 in blocking solution for 1-2 hr on a rocker at RT. Then, the membranes
were washed with PBST three times as above. After the final wash, the membranes were
incubated with 6 ml secondary antibodies conjugated with horseradish peroxidase in
1:1500 dilution on a rocker at room temperature for 1 hr and then washed extensively
with PBST. Chemiluminescent detection (ECL, Amersham) was used to reveal the
specific immuno-conjugated protein for Western blot. The membrane was covered with 2
ml ECL detection reagent for 1 minute at room temperature (1 ml of substrate and 1 ml of
buffer were mixed in a centrifuge tube). Membranes were than placed in a film cassette

and exposured for 30 seconds to 3 minutes.

23

5. Flow cytometry

The expression of p53 in clones with extended lifespan was studied by Western blot
for quantitative analysis at the protein level and by flow cytometry for functional analysis
ofp53.

a. X—ray irradiation and fixation of cells

Cells were cultured in three 100 mm dishes until 70 % confluent. One dish was used
as control, the others were irradiated with X-rays for 4 Gy and 8 Gy at dose rate of 2 Gy
/min. The medium was replaced immediately after irradiation. After 24 hr incubation,
cells were rinsed with PBS once and removed by trypsinization for subculture. Cells were
counted and aliquoted 2x106 cell per centrifuge tube. To remove trypsin, cells were
pelleted, washed with PBS once and pelleted again. Then 2 ml of cold 70% ethanol was
added to fix cells for 1-3 hr at 4°C. Cells were stored at -20 0C until staining.

b. DNA staining and DNA content analysis

The ethanol -fixed cells were centrifuged at 1000 rpm at 4°C for 5 minutes. The
ethanol was removed as much as possible by blotting on tissue papers. The cells were re-
suspended in 3 ml PBS, transferred into a 12x75 mm tube and centrifuged as above. After
decanting the PBS, the cells were re-suspended in 0.5 ml DNA staining reagent (0.1%
triton X-100, 0.1 mM EDTA, 0.05 mg/ml RNaseA and 50 pg/ml propidium iodide in
PBS, pH7.4) and incubated overnight in dark at 4°C. The cells were analyzed with a

fluorescence-activated cell sorter (FACS) (Becton Dickinson) available in the Dept. of

Biochemistry, Mich. State Univ.

24
Materials

The sources of chemicals, antibodies, cell culture supplies and reagents are listed as

 

 

follows.
Chemicals:
Sodium Dodecyl Sulfate(SDS) Boehringer Mannheim,
Glycine Indianapolis IN
PMSF Sigma, St Louis MS

Bovine Serum albumin, EDTA
Propidium iodide, Ponceaus reagent

B-mercaptoethanol, Bromophenol blue

 

30 % acrylamide, TRIS, TEMED BIO-RAD, Hercules CA
Ammonium persulfate, Tween 20

DC Protein assay, Triton X-100

 

 

 

 

 

Cello-seal grease Fisher, Fair Lawn NJ
PVDF membrane Millipore, Bedford MA
Methanol J.T. Baker, Phillipsburg NJ
ECL detection reagent Amersham Life Science,

Arlington Heights, IL

 

 

 

25

Cell culture
Normal human breast epithelial cells (HBEC) derived from reduction mammoplasty of
different women were used. The cells used in this study were Human Mammary
Epithelium (HME): HME 5, HME7, HME12, HME14, HME15, HME17 and HME20.
The extended lifespan clones were derived from two HBEC cultures after X-ray
irradiation (A designated initial plate, B designated second plate).
M15XA6, M15XA12 ------ HME15A (20 Gy)
MlSXBS, M15XB6, M15XB7, M15XB11, M15XBl2 ------ HMEISB (12Gy)
M15XA25L1, M15XA25L4, M15XA25L7 ------ HME15A (80y)
M15XB23L2, M15XB23L3, M15XB23L4, MlSXBZSLl ------ HMEISB (10Gy)
M20LBX1, M20LB6-3 ------ HME20 (10Gy)
M12B4 ------ an extended life clone derived from HME12 after 5-bromodeoxyuridine
(BrdU) treatment was also used in the study.
The E.L. clones used for experiments were at 25-35 cpdl. Except three clones from
M15XBZ3 which were recovered from the same plate, all others are independent clones
from different dishes.
Some immortal and cancer cell lines were also used :
M13SV22-—-- HME13 Type I HBEC immortalized by SV40
MISSV30---- HME15 Type II HBEC immortalized by SV40
M13SV1R2N1---- HME13 Type I HBEC immortal and tumorigenic after SV40,X-ray
and neu transformation
MCF-7---- breast cancer cell line

T47D---- breast cancer cell line

Medium /growth factors/antibodies

26

 

Bovine pituitary whole

Pel-Freez, Rogers AR

 

Fetal bovine serum
Trypsin

Gentamicin

Life Technologies,

Gaithersburg MD

 

MSU-l medium, 17-13 estradiol

Insulin, Human transferrin
Hydrocortisone,

Epithelium Growth Factor

Sigma, St Louis MS

 

P53 (Ab-2) pAb1801

Rb (Ab-5) LM95.1

Calbiochem-Novabiochem

International, Cambridge MA

 

P16 (C20) a.a.l37-156
P21 (C19) a.a.]46-164

Cyclin D1 (HDl 1)

Santa Cruz Biotechnology,

Santa Cruz, CA

 

Anti rabbit IgG horseradish peroxidase

linked whole antibody

Amersham Life Science,

Arlington Heights, IL

 

 

Anti mouse IgG horseradish peroxidase

linked whole antibody

 

Amersham Life Science,

Arlington Heights, IL

 

 

27

RESULTS

1. Induction and isolation of extended lifespan (E. L.) clones from Type II HBEC

after X-ray irradiation

Ten experiments were carried out to induce extended lifespan of Type 11 normal
HBEC by X-ray irradiation as listed in Table 1. In seven of these experiments, duplicate
plates were used (designated as A and B plate in Tablel). In these experiments, Type II
HBEC derived from reduction mammoplasty of six different women were used (i.e.
HMES, 7, 12, 14, 15, 20). The cells were exposed to multiple (3-6 times) low dose (2 or 4
Gy) X-rays for a cumulative total dose of 6-20 Gy. These experiments yielded twenty-six
putative E. L. clones from two different cell subjects (HME15 and HME20) in three
successful experiments. To confirm if the isolated putative E. L. clones are indeed having
extended lifespan according to our definition (i.e. 24 cpdl), the cpdl of each of these
clones was determined. The results as listed in Table 2 shows that twenty-four of these
clones are indeed E. L. clones. Normal Type II HBEC (Fig 2.)were highly proliferative in
early stage and became senescent (Fig 3.) within two months. In these experiments, most
cells were proliferating actively in early stage until they became senescent when they
changed cell morphology (enlarged) and stopped proliferating. At this stage, a small
fraction of cells may continue to proliferate and form E. L. colonies (Fig 4, 5.). The
morphologies of early passage E. L. clones are indistinguishable from early passage
HBEC (Fig 6.). In late passage, these E. L. clones also became senescent showing some
elongated fibroblast -like cells or giant multinuclear cells (Fig.7).
Wazer et al. (1994) have reported the isolation of an immortal HBEC clone after

irradiation of cells with a relatively high cumulative dose of X-rays (30 Gy).

28

 

Fig 2. The morphology of normal Type H HBEC (HME20 ) cells.
( phase contrast,100X)

 

Fig 3. The morphology of senescent Type II HBEC (HME15 ).
The small and pack cells are healthy cells, the large cells are senescent cells.
(phase contrast 100X)

29

 

Fig 4. A colony proliferating Type II HBEC after 6 Gy X-ray exposure.
(phase contrast, 40X)

 

Fig 5. An extended lifespan colony proliferating after X-ray irradiation.
(M15XA25) (phase contrast 40X)

 

Figure 6. Morphology of the E. L. cells in early passage.

A. M15XA25L1 B. M15XBZ3L3 C. M20LBX1
Cells were at 30 cpdl (phase contrast 100X)

.1

31

 

Figure 7. Morphology of the E. L. cells in late passage.(phase contrast 100 X)
A. M15XB23L4 showed enlarged and multinuclear cells at about 45cpdl.
B. M15XBZSL1 appeared fibroblast-like at 50 cpdl.
C. M20LBX1 showed the giant and multinuclear cells in about 36 cpdl.

32

 

Figure 8. The E. L. clone after 20 Gy X-ray irradiation

M15XBZSL1 showed multinuclear and large cell morphology (phase
contrast 100X)

To determine if continual exposure of our E. L. clones could convert them into immortal
clones, eight highly proliferative E. L. clones (cpdl 25-35) were irradiated again with X-
rays (2 or 4 Gy) each time for an additional total dose of 8 or 10 Gy (Table 3.). The total
cumulative doses for these clones range from 20-28 Gy. All of these cultures, however,

eventually became senescent (Fig 8.) and no immortal clone emerged.

33

Table 1. Induction and isolation of extended lifespan (E. L.) clones from
Type II HBEC after X-ray irradiation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Exp. Date cells Treatment Total dose Extended life
(dose x times) (Gy) clone obtained
1 7/10/95 HME15A 4 x 5 20 Zelones
HMEISB 2 x 6 12 6clones
2 12/6/95 HMEISA 2 x 4 8 11 clones
HMEISB 2 x 5 10 Sclones
3 2/29/96 HME14A 2 x 3 6 None
HME14B 2 x 3 6 None
4 4/20/96 HME14B 2 x 3 6 None
5 5/20/96 HMESA 2 x 4 8 None
HMESB 2 x 4 8 None
6 5/20/96 HME7A 2 x 5 10 None
HME7B 2 x 5 10 None
7 7/31/96 HME12A 2 x 4 8 None
HME12B 2 x 4 8 None
8 7/31/96 HME14A 4+2x3 10 None
HME14B 4+2x3 10 None
9 8/20/96 HME20R 2 x 3 6 None
10 8/20/96 HME20L 2 x 5 10 2clones

 

 

 

 

 

 

 

 

34

Table 2-1. The proliferation potential of putative E. L. clones isolated
from Type II HBEC (HMEIS) after X-ray irradiation
The cpdl of BL. clones. Cells were trypsinized and counted on the indicated date .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DATE 9/8/95 9/18/95 9/29/95 10/12/95
M15XA6 21.9 26.5 29.8 32.9

DATE 8/22/95 9/5/95

M15XA12 23 26.8

DATE 8/30/95 9/1 1/95 9/25/95

M15XB3 21.8 24.6 27.8

DATE 8/27/ 95 9/8/95

M15XB5 22.3 26.7

DATE 9/ 1 3/ 95 9/25/95 10/18/95

M15XB6 22.1 27 30.3

DATE 8/25/95 9/8/95

M15XB7 21.5 24.7

DATE 9/5/95 9/17/95 9/29/95 10/10/95 1 1/6/95
M15XBll 22.3 27.2 31.2 33.5 36.8
DATE 9/6/95 9/17/95 9/29/95 10/12/95
M15XBlZ 22.5 25.6 30.3 32.1

 

 

35

Table 2-2. The proliferation potential of putative E. L. clones isolated
from Type II HBEC (HME15) after X-ray irradiation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DATE 3/15/96 4/11/96

XA23L1 21.3 25.6

DATE 3/12/96

XA23L7 22.3

DATE 2/5/96 3/6/96 3/25/96

XA25L1 23.1 25.8 30.7

DATE 2/5/96 3/12/96 3/29/96

XA25L2 24.4 27.5 33

DATE 2/5/96 3/12/96 4/9/96

XA25L3 23.4 26.9 29.7

DATE 2/5/96 3/6/96 4/3/96

XA25L4 22.5 26.4 30.2

DATE 3/25/96

XA25L5 20

DATE 2/5/96 3/ 13/96 4/9/96

XA25L6 23.4 26.9 30.8

DATE 2/5/96 3/6/96 3/25/96

XA25L7 23.1 26.6 31.5

DATE 2/5/96 3/ 13/96 4/3/96

XA25L8 23.3 27.2 31.3

DATE 2/5/96 3/ 13/96 4/30/96

XA25L9 22.7 26.8 29.9

DATE 3/22/96 4/5/96 4/ 17/96 4/29/96 5/13/96 5/31/96
XBZ3L2 22.1 26 30.8 34.6 39 42.5

DATE 3/21/96 4/2/96 4/17/96 4/29/96 6/8/96 7/5/96
XB23L3 23.7 29.2 35.2 39.9 43.7 47.2

DATE 3/15/96 3/29/96 4/16/96 5/2/96 5/21/96 6/8/96 7/5/96
XBZ3L4 21.8 27.2 33 37.8 42.5 46.6 48.5
DATE 3/15/96 3/26/96 4/11/96

XB23L5 22.3 27.2 33

DATE 3/12/96 3/25/96 4/8/96 4/19/96 4/29/96 5/8/96 5/20/96 6/8/97
XBZSLI 23.4 29.3 35.1 38.1 40.6 44.7 49.1 52.2

 

 

36

Table 2-3. The proliferation potential of putative E. L. clones isolated
from Type II HBEC (HME20) after X—ray irradiation

 

 

 

 

 

DATE 12/6/96 12/23/96 1/2/97 1/13/97 2/3/97
M20LBX1 22.4 25.5 30.6 34.9 38.6
DATE 12/16/96 2/ 1 0/ 97

M20LB6-3 23.4 27.8

 

 

 

 

 

 

 

Table 3. Results of additional X-ray irradiation of E. L. clones

E. L. cells at cpdl 25-35 were exposed to X-ray at 4Gy /2Gy the addtional exposure, total
accumulated dose and result are listed.

 

 

 

 

 

 

 

 

 

 

 

 

 

CELL ADDITIONAL TOTAL EXPOSURE Final outcome of
EXPOSURE cell culture
M15XA6 +8 GL 28Gy Senescence
M15XB11 +10 Gy 22Gy(XB11 22 Gy) Senescence
M15XBZ3L2 +10 Gy 20Gy (XB23L2 20Gy) Senescence
M15XBZ3L3 +10 Gy 20Gy (XB23L3 20Gy) Senescence
M15XB23L4 +10 Gy 20Gy (XB23L4 20Gy) Senescence
M15XBZSL1 +10Gy 20Gy (XB25L2 20Gy) Senescence
M20LBX1 +10 Gy 20Gy (LBXl 20Gy) Senescence
M20L6-3 +10 Q‘L 20Gy (20LBX2) Senescence

 

 

37

2. Failure to obtain E. L. clones from Type I HBEC after X-ray irradiation
Both Type I and Type II HBEC were obtained from first passage of each cell culture.
Type II HBEC were used in experiments described previously. The number of Type I
cells is usually considerably less than Type 11 cells. The colony- forming cells derived
from each vial are estimated to be about 500 and 50,000, respectively for Type I and
Type II HBEC. After culturing 4-5 days for Type II HBEC and 10 days for Type I
HBEC, these Type II and Type I cells attaining a total of about 2 x 106 and 2 x 105 cells
respectively were irradiated to initiate the experiments. The experiments carried out to
induce E. L. clones from Type I HBEC by X-rays are listed in Table 4. In initial
experiments, it was noticed that Type I HBEC were more sensitive to low-dose X-ray
treatment (1-2 Gy). Very few colonies were formed after the subculture of these
irradiated cells. Therefore, the doses of X-rays in each treatment were decreased in latter
experiments (0.1-0.6 Gy). The reduction in dose allowed the cells to have prolonged
proliferative activity. After repeated treatment, the cells eventually became senescent. No

E. L. clone was found from these experiments (Table 4.).

38

Table 4. Results of the effects of repeated X-ray irradiation of

Type I HBEC on induction of E. L. clones

Cells derived from different subjects and the treatments are listed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cells Treatment (Gy) E. L. clone obtained

1 HME15 2,2,2 (2Gy/min 1min) None
2 HME15 1,1,1 (2Gy/min 30 sec) None
3 HME15 1,1 None
4 HME15 .5,.5,.5 (2Gy/min lSsec) None
5 HME14 .5,.5 None
6 HME15 .5,.5 None
7 HME17 .5,.5 None
8 HME15 2,2,2 None

1,1,1 None

1,1,1 (O.SGy/min 2 min) None
9 HME14 .l,.3 (0.2Gy/min 303ec) None
10 HME5,7 .1,.3,.6,.5,.5 None
11 HME12 .1,.3,.6,1,2 None
12 HME14 .1,],l,1,l None
13 HME15 .1, 1,1,2,2,2,2 None
14 HME15 .3,.3,.3,.3,.3,.3,2,4,4, None

 

 

 

 

 

39

3.Expression of genes related to cell cycle regulation

The genetic and molecular basis for lifespan extension in HBEC induced by X-ray
irradiation is not clear. To gain insight into the mechanism of the first significant change
related to transformation (i.e. lifespan extension), the E. L. clones isolated were
characterized for the expression of genes related to cell cycle regulation.

A.p53
Western blot analysis

The p53 protein expression was studied by Western blot analysis. Cells defective in
p53 may show decreased or enhanced amounts of p53 protein. The latter is attributed to
the longer half-life of some mutant p53 protein compared to the wild-type p53. In
experiments to determine the p53 protein expression in E. L. clones (F ig.9-10), two
breast cancer cell lines, MCF-7 and T47D, which express wild-type and mutant p53,
respectively, were included in the experiments as shown in Fig. 9. T47D cells contain
considerably more p53 proteins than MCF-7. Also included in the experiments are two
SV40 immortalized Type II HBEC (M13SV22 and MlSSV30) and one tumorigenic SV
40 immortalized Type I HBEC (M13SV1R2N1). The high level of p53 proteins
expressed in these cells are presumably inactivated and stabilized by complexing with the
SV40 large T antigen. Thirteen E. L. clones isolated from HME15 were examined in the
Western blot analysis. In two of these clones, cells at different stages of X-ray irradiation
were studied (i.e. XBl 1, XBll 22 Gy; XB25L1, XB25L1 20 Gy). The results (Fig. 9)
show that seven of the thirteen clones appear to contain elevated level of p53 protein
(XA25L7, X81 1, XB12, XB23L2, XB23L3, XB23L4, XBZSLI) compared to the

parental Type II HBEC (HME15).

40

 

 

A.
E
3
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g _. xx ..a
can): go:
”garage 883222
manua- ~13: 5:93;,3
B. 33
m
f."
XXX -‘
SEE xxeasza
as Eagméaawaaas
U303 N‘§:N‘-¥QSI:’w-¥‘<O
P53
cyD
C.

3
d
u
m
<
A
”
N
Z
.3

V9 L
L‘ISZVX
3 E‘IEZSX
V'IEZQX
L'ISZSX

     

x
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N
a:
r
a

, max
,gz1ezax

P53

Figure 9. p53 expression in E. L. clones by Western blot

Clones are listed on top. Normal Type II HBEC (ISB, 15A),
positive controls (T47D, MCF-7, M13SV22, MISSV30, and
M13SV1R2N1). In between of normal and positive control are E.L.
clones induced by X-ray irradiation or by BrdU treatment (M12B4).
CYD: cyclin D1

41
E. L. clones at different stage of X-ray irradiation may exhibit different level of p53
protein as shown by XBZSLI. An E. L. Type II HBEC clone transformed by 5-
bromodeoxyurindine (BrdU) in a previous experiment (M 12B4) also contain higher
amount of p53 protein. All the E. L. clones derived from HME20 also contain higher
level of p53 protein (Fig.10). In this experiment, the nuclear and cytoplasmic fractions of
cell lysate in two clones were separately assayed. It is clear that the p53 proteins are

located in the nucleus in these cells.

EN 903 IvXS'IOZW

OSASS WI

”(8102“
MO 902 txa'iozw

i.

nozaozw
‘ auzaozw

‘ e-sa‘lozw

ji ZXG‘IOZW
3N ocAssm
no ocAssm

   

I, .1 81on
r

Figure 10. Expression and localization of p53 in HME20 normal
and E. L. cells studied by western blot.

Experiments using different fractions of cell extracts: Normal Type II
HBEC total protein (M20LB), cytoplasmic fraction (M20R2CYT),
and nuclear fraction (M20R2NE); the positive control total protein
(M158V30), cytoplasmic fraction (M158V30 CYT), and nuclear
fraction (MISSV3ONE). Position of p53 proteins are indicated by
arrow.

42

Flow cytometry analysis

To determine whether the increase in p53 protein is due to the presence of mutant
p53 or not, a firnctional p53 assay was done for six of these E. L. clones. Cells with wild
type p53 normally show G1 arrest after exposure to ionizing radiation. Cells with mutant
p53 show only 02 arrest instead of GI arrest. The status of cell cycle is reflected by cells
with different DNA content which can be detected by a flow cytometer. The data of
F ACS were shown by charts with cell number (Y-axis) vs. DNA content (X-axis). The
first peak is G1 phase and the second peak at twice the DNA content is G2 phase, and the
broad distribution between the two peaks is the S phase. The results of these studies are
summarized in Table 5. Overall, these results did not provide evidence that the E. L.
clones examined are defective in p53 function since no X-ray-induced G1 arrest was
evident.

MCF-7, a breast cancer cell line which expresses wild type p53, was chosen as a
positive control. Afier 4 Gy exposure, the cells in G1 increased to 91.1% of total cells
from 71.4% in non-irradiated cells (Fig 11). The results of MCF-7 show a clear G1 arrest
after X-ray irradiation. The two negative controls are T47D and M158V30. The former is
a breast cancer cell line with mutant p53 and the latter is a Type II HBEC (HME15)
immortalized by SV40. These cells show a dramatic decrease cells in G1; a decrease
from 61.7% to 40.8% for MISSV30 and 66.8% to 33.6% for T47D (Fig.12, 13). In the
meantime, cells were arrested in G2. The percentage of cells in G2 increased from 5.3 to
28.6 for T47D and from 9.0 to 44.6 for MISSV30. After 8 Gy irradiation, G2 peaks are
higher than 01 peaks in T47D and MlSSV30 (data not shown). For parental Type II

HBEC 53% of cells remain in G1 after exposure to 4 Gy of X-ray irradiation (Fig. 14)

43

compared to 58.6% of non-irradiated cell, indicating radiation induced G1 arrest. The
percentage of G1 cells in XA25 decreased to 47.1 from 63.3 after 4 Gy X-ray irradiation
(Fig. 15). Similar results were shown in the XB25L1 with 56.9% in control and 49% for
4 Gy X-ray irradiated cells (Fig. 16). Although with a lower p53 and p21 expression
(results to be shown), the XB25L1 20 Gy clone also showed a similar effect of G1 arrest.
(45.8% in G1 after irradiation compared to 54.6 % for non-irradiated cells) (Fig.17). The
HME20, the normal Type 11 cells showed a significant G1 arrest after irradiation. The
percentage of G1 cell increased to 87 after 4 Gy X-rays exposure, while the control only
has 71.3 % of cells in G1 (Fig.18). The extended lifespan clone from HME20, 20LBX1,
showed a pattern similar to HME15 and HME15 E. L. clones and slightly different from
its parental cells, i.e. the percentage of G1 in irradiated cells slightly lower than the non-

irradiated cells (59.2% vs.64.6%) (Fig.19).

Table 5. Summary of results of cell-cycle analysis for
control and X-ray irradiated E. L. clones
Cell cycle date were analyzed with a flow cytometer. Cells and treatments are

listed in the first column. The percentage of cell in different cell cycle stage are

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

shown .

CELLS Gl S G2
15B 58.6 32 9.5
15B-2 65.1 26 9
153+ 4Gy 48.6 28.7 22.7
153+ 4Gy#2 53 14.7 32.3
15B+ 8Gy 40.6 18.4 41
15XA25pool 63.3 16.6 20.1
15XA25pool+4Gy 47.1 18.8 34
15XA25pool+8Gy 47.7 1 1.9 40.4
15XBZ3L2 58.8 16.9 24.3
15XBZ3L2 +4Gy 57.3 11.9 30.7
15XB23L2 +8Gy 60 10.8 29
15XB23L4 64.7 20.6 14.7
15XB23L4+4Gy 50.7 23 26.3
15XBZ3L4+8Gy 44.5 31.3 24.2
15XBZSL1 56.9 18.4 24.7
15X825Ll+4Gy 49 23.6 27.4
15XB25L1+8Gy 44.5 13.7 41.8
15XB25L1 ZOGy 54.6 8.1 37.2

15X825L1 20Gy+4Gy 45.8 27.3 26.9

15XB25L1 20Gy+8Gy 50.3 13.3 36.5

 

 

45
Table 5 (Cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cell G] S G2
20LB 71.3 26.3 2.5
20LB +4Gy 87 2.6 10.4
20LB +8Gy 75.9 20.2 3.8
20LBX1 80% confluence 81.1 6.5 12.4
20LBX1 density arrest 88.4 1.9 9.7
20LBX1 64.6 17.5 17.9
20LBX1 +4Gy 59.2 12.1 28.7
20LBX1 +8Gy 47.7 17.1 35.2
MISSV30 61.7 29.3 9
M158V30 +4Gy 40.8 14.6 44.6
MlSSV30 +8Gy 17.4 10.4 72.2
T47D 66.8 27.8 5.3
T47D +4Gy 33.8 37.6 28.6
T47D+ 8Gy 29 28.7 42.2
MCF-7 71.4 25.6 3
MCF7 +4Gy 91.1 8.6 0.3
MCF7+ 8Gy 92.8 2 5.2

 

 

 

 

 

46

atasrnz new c1 na-a

 

480.

400.

320.

BOG,

150.

Cell Number

30.

 

82

 

DNA Content

AUIHEBMV7X1 ”31

 

 

 

49W.

400.

380.

340.

160.

Cell Number

80.

 

 

 

' :' c 36 5%
DNA Content

 

113m

Mean 01
CV G1
% G1
Mean G2
CV GZ
% G2

% S
G2/G1
Chi Sq.

Mean G1
CV Gl
% G1
Mean 62
CV G2
% G2

% S
G2/G1
Chi Sq.

= 27.9
= 6.2
= 71.4

53.4
6.0
3.0

= 25.6
= 1.917

1.3

= 29.2
= 6.1

= 91.1
= 56.8

6.8

.3

8.6
1.946
1.4

Fig 11. Cell cycle—MCF-7 A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The 01 peak in irradiated cells is

higher than that in control group.

47

00:32:47 m c: I'm-4L

 

 

 

 

 

 

A. 146 f
read.
8 icon. %Gl = 61.7
2 non. .. Mean G2 = 88'6
300. ‘r
d
(3 «on. 1' %S = 29.3
300. GZ/Gl = 1.919
Chi Sq- = 1'7
q — _ :uh z I ‘- I
DNA Conteat
"human” rue-a
B. 76 I - ' ' v 6 :
Mean G1 = 46.9
son.
3 son. %G1 = 40.8
2 400. MeanGZ = 900
Z and.
—. %G2 = 44.6
a 800, 1, %S = 14.6
100. ,_ GZ/Gl = 1.919
‘ . ‘ Chi Sq. = 1.5
. ”/17. /, I- 3:7z// "“ .

 

 

DNA Content

Fig 12. Cell cycle-M15SV30 A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped

while G2 peak increased.

48

31.861735 T470 C 71.3-A

 

A. 140

1800.

1008»

8001.

5001.

400.

Cell Number

300..

 

si

 

‘. I. ...I .“'.— -

‘v

5‘ T 27 ”757.71.? /
’4.//.4///A/// ‘

DNA Content

 

M173 7170 XI [1.3-0

 

  

 

soul

Cell Number

4001,

2001»

 

Tsz'

 

DNA Content

 

Mean G1
CV G1
% G1
Mean G2
CV G2
% G2

% S
G2/Gl
Chi Sq.

Mean G1
CV Gl
% 61
Mean G2
CV G2
% G2

%8

G2/G1
Chi Sq.

= 31.4
= 6.1
= 66.8
= 60.3
= 4.9
= 5.3
= 27.8
= 1.921
= 3.3

29.3
7.4

= 33.8
56.6
7.0
28.6
37.6
1.930
= 3.5

ll

Fig 13. Cell cycle—T47D A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped

dramaticly while G2 peak increased.

49

M.“ 1701 15' 513-0

 

A. 160

   
 

      
 

 

 

 
           

 

  

'2: ‘5 Mean G1 = 35.5
I ' cv G1 = 3.9
.. “3°“ l: %G1 = 58.6
E I, Mean oz = 69.2
:s 8001. 1. cv G2 = 4.6
E 3 %G2 = 9.5
3 4m T %s = 32.0
" GZ/Gl = 1.947
DNA Content
3. .26 PM"? “"1“ '2 - "3". -
» ‘2 Mean G1 = 31.7
100d. CV G1 = 5.5
.. %Gl = 53.0
0 son.
.0 Mean G2 = 59.7
25 son. 1 CVG2 = 5.7
d %02 -—= 32.3
3 “"3 %s = 14.7
m, G2/Gl = 1.886
Chi Sq. = 2.7

 

 

 
   

DNA. Content

Fig 14. Cell cycle—HMEISB A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped
while G2 peak increased.

50

01.517“ lSXAZS 71.3-A

 

 

A. 200 . - ,,
Mean G1 = 31.9

.566. ‘ , cv G1 = 4.8

33 1; %G1 = 63.3
E .2611 g MeanG2 = 60.1
2 h cv oz = 5.4
3 8991 H %(}2 = 20.1
0’ 1 %s = 16.6
“’01 ‘ ‘1 GZ/Gl = 1.881

If Chi Sq. = 1.8

 

 

  

 

DNA Content

AL“ l7" ISXAZS X1 71.3-0

 

B. 120 - . 82
Mean G1 = 31.2
1000.. CVGI = 60
‘6 arm. %Gl = 47.1
2 Mean G2 = 59.4
:2 6°“: CV G2 = 6,3
—t 4001,
{3 %s = 18.8
aoq, 62/61 = 1.901
Chi Sq. = 1.1

 

 

DN.A Content

Fig 15. Cell cycle—M15XA25 A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped

while G2 peak increased.

51

81.061716 lSXlZS 1.1 ru-n

 

A. 1410
race,

a roan.

0

.0

E son.

:1

2 son,

H

.4

6 «on.
and.

 

“1717 15!“ L1 X1 [1.3-0

.. ’ .,, gill):
r . I, .
,/ . If
I ’/v ' / ' ~
I///. 711/” . . ,r ‘

Si

 

DN.A Content -

 

 

 

B. 180

10001.

Cell Number

3001.

BOG.

3001.

4OQ.

 

.3
!

  

.-..‘

. I “. .
t? 1' / ‘. .i
'3 ‘f, - ":1
r , ' / . .
. 4291/1“. ~

DN.‘\ Content

 

Mean G1
CV Gl
% G1
Mean GZ
CV G2
% G2

% S
G2/Gl
Chi Sq.

Mean G1
CV G1
% G1
Mean G2
CV G2
% G2

% S
G2/G1
Chi Sq.

29.9
6.1
56.9
58.3
7.0
24.7
18.4
1.950

29.0
6.2
49.0
56.6
6.8
27.4
23.6
1.953
1.7

Fig 16. Cell cycle-M15XB25L1 A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped

while G2 peak increased.

52

ALMI'MS lSXIZS L1 286 FIJI-A

 

A0 130

10001,

300..

300i

40:11

Cell Number

80011

 

c/ o ' “ u; i "
. .- a ,‘r 1.’ 1, ‘r 1,
W71 r. 7' [#01:—

DNA Content

  

f

82

fimlm 1m L1 2“ X1 71.3-A

 

 

 

B. 120

10001,

800.

500..

4001,

Cell Number

8001.

 

 

V

82

DNA Content

 

Mean G1
CV G1
% G1
Mean G2
CV G2
% G2

% S
G2/Gl
Chi Sq.

Mean G1
CV G1
% G1
Mean G2
CV G2
% G2

% S
G2/G1
Chi Sq.

31.1
6.8
54.6
58.8
8.8
37.2
8.1
1.888
1.3

29.8
6.8
45.8
57.6
8.0
26.9
27.3
1.933
2.1

Fig 17. Cell cycle-MlSXBZSLl 20 Gy A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped

while G2 peak increased.

53

 

amrnq zou c1 ru-a
A, 160 - _' ' ' ' 32'

1200..

80Q1

Cell Number

400.

  

 

 

DNA Content

01.061725 '20:.- 4611 m-A
' I T ' si

 

Bo 200

16001.

    

1200i.

800..

-- 1' ! "799-! ..p. 3‘
5:3, {454.44 1:11:38. - '

Cell Number

4001.

 

 

DNA Content

Mean G1
CV G1
% G1
Mean G2
CV 62
% G2

% S
G2/G1
Chi Sq.

Mean G1

CV G1
%G1

Mean G2

CV G2
% G2
% S
G2/G1
Chi Sq.

31.2
6.3
71.3
62.3
8.3
2.5
26.3
1.996
3.4

= 31.6
= 6.4
= 87.0
= 60.0
= 8.1
= 10.4
= 2.6
= 1.899
= 1.3

Fig 18. Cell cycle-HME 20 LB A: control, B: 4 Gy irradiation
DNA content was measured by a flow cytometer. The G1 peak in irradiated cells is

higher than that in control group..

54

“L06 1729 20LBX1 C 113-3

 

 

 

 

 

 

A. aoo . 82
Mean or = 30.9
16001. : , CV G1 = 5.3
u f °/oGl = 64.6
0) ,
E 1" Mean G2 = 60.4
=1 CV G2 = 5.9
.2. %G2 = 17.9
8 %S = 17.5
mm = 1.957
Chi Sq. = 1.5
“17320118146? [1.3-A
BO 140 e' ' ‘. . 82 .
1! ~ Mean o1 = 30.5
.26... 1 ,
11 CV o1 = 5.7
3 "m %Gl = 59.2
E 809. MeanG2 = 59.3
5 CV G2 = 5.8
2 ...... 1
3 “ %G2 = 28.7
0’ “‘1' 1, ,,- 3 %s = 12.1
.ée- 1’1?
200. 1,; mm = 1.947
.1 $3,," . Chi Sq. = 1.7

 

 

DNA Content

Fig 19. Cell cycle—20LBX1 A: control, B: 4 Gy irradiation

DNA content was measured by a flow cytometer. The G1 peak in irradiated cells dropped
while G2 peak increased.

55
B. p21

Induction of p21 after irradiation requires functional p53. MISSV30, which
presumably contains the inactivated p53, showed only low level of p21 expression (Fig
20). There is no or trace expression of p21 protein in T47D and MISSV30, consistent
with the presence of inactivated p53 in these cells. Both parental normal Type 11 cells
(HME15 and HME20) expressed small amount of p21. In contrast, most extended
lifespan clones showed an increase in the expression level of p21. In total, fifteen and
five E. L. clones (or its sub-populations receiving different dose of X-rays) from HME15
(Fig. 20, 21) and HME20 (Fig. 22) were analyzed. In the HME 15 E. L. series, the p21
proteins in ten clones were clearly elevated (Fig. 20, 21). For HME20 E. L. clones, the
p21 proteins were greatly elevated in four of the five clones tested (Fig. 22). A clone at
different stage of the X-ray irradiation may show great difference in p21 expression (i.e.
high expression in XBZ3L4 and low expression in XB23L4 20 Gy)(Fig. 20). The p21
proteins, similar to p53, were primarily found in the nuclear fraction as shown in one
clone studied (Fig. 22). The expression level of p21 is also correlated with the level of
p53 in extended life cells. Clones that highly expressed the p53 were also found to
contain relatively high level of p21 expression (Fig. 23). The chart is from the Fig. 9a.
and Fig. 21. The two gels were run at the same time using same samples. After
completion of Western blot and chemifluorescent detection, the films were scanned into a
computer and the density of the bands was analyzed by Sigma gel program. The
concomitant expression of high level of both protein suggests that p53 in extended life

clones is functional.

56
C. Cyclin D1

The cyclin D1 proteins are expressed at relatively high levels in both parental Type
11 cells (HME15 AND HME20) compared to that in MCF-7 and T47D cancer cell lines
or in the SV40 immortalized Type 11 cell line (MISSV30) (Fig. 24, 25). Except for A
few clones (i.e. XA6, XA25L1, XA25L4, and XA25L7), most of the E. L. clones tested

(total fifteen) are not greatly different from the parental cells in cyclin D1 expression.

NS
0101
l_|"'
fifl

,Z'Iezax
mezax

- v1czax

, ooz-v1ezax
i HSZSX
"jooz-nszax
osAssm

LEX

    

Figure 20. P21 expression in E. L. clones derived from HME15

Normal Type II HBEC (M15B), immortal Type II HBEC (M15SV30),
and the E. L. clones of HME15 were studied by Western blot.

g
—I
§ 52 §§
W 01
§§E§§iégég
§°~§~>°25=e><aaszcg>
agingnnmofii—r-gfl'."
WON.Nmfl<—‘Nh _U\‘

f
i

 

Figure 21. p21 expression in E.L. clones

Normal Type II HBEC (M15B), breast cancer cells (T47D, MCF-7), E.L. from
HME15 by x-ray and EL. from HME12 by chemical(MIZB4) were studied by
Western blot.

no 11902 lXQ‘lOZW
3N OSASQLW
no OSASSLW

81031111
MOZHOZW
BNZHOZW
998103101
ZXB'IOZW

lXG'lOZlN

OSASS 1W

P21 .3

. EN 1190?. rxenozw

Figure 22. p21 expression in E. L. clones derived from HME20
Different cells fractions were studied: Normal Type 11 HBEC total protein
(M20LB), cytoplasmic fraction (M20R2CYT), and nuclear fraction
(M20R2NE). the positive control total protein (M15SV30), cytoplasmic
fraction (M15SV30 CYT), and nuclear fraction (M15SV30NE).

 

p53 921

 

 

relative
ratio

 

 

N
,_
<
><

XA25L7

 

 

 

Fig 23. Co-expression of p53 and p21 in normal and EL. cells.

Data are from Fig 9A. and Fig 21. Two western blots were scanned into a
computer and the density of the bands was analyzed by Sigma gel program.
The relative ratio is set as multiples of control (M l 5B=1).

 

B. 3
xx -‘
3 § Emequ
ignxannnn g
a...“ mo. w<nw11<~I
mNNUINnh-b °
-..... -----

Figure 24. CyclinDl expression in E. L. clones

Normal Type II HBEC (M15B) ,two breast cancer cells (T47D,MCF-7),
immortal Type II HBEC (M15SV30 ) and EL. clones in between.

E g
o N
[— O
a: E g g
x >< -* a
Z 3 z z z E; -‘ z 8 m
'3 B N N N o g .. < 5
Z 21 DO 19 o o G) o "' °° o
N N M w '— 1— < w o
C) O 03 ED 0 '< < Z o
w 1’; n1 co -* j 111 O

cyclin D1 .

 

Figure 25. Expression and localization of cyclin D1 in E. L. clone
derived from HME20
CYT : cytoplasmic fraction; NE: nuclear fraction, others are total protein

59
D. RB

The molecular weight of pr is about 105-110Kda; the amount of these two
different molecular forms depends on the status of phosphorylation. The phosphorylated
form is inactive and can be found in proliferating cells. There appears no significant
change in total amount of Rb between E. L. clones and their parental normal cells.
However, there is a difference in phosphorylation of Rb (Fig. 26, 27). There is only one
upper band (hyperphosphorylated form) in normal Type 11 cells ( 15B) and in the
immortal cell line (M15SV30). All except one (XB7) of the E. L. clones showed two
bands of Rh, representing hyper and unphosphorylated form. XB7 only has

hyperphosphorylated band (Fig. 27).

 

X x

g m
u g 3
§§ 855*58333
3 ”NXNNNS ”1"an
z: mamegam°§3”<
mo5.:ua~<59_.g.fig
th| ;

r
1

'I—l'b-

 

1..

 

Figure 26. Rb expression and phosphorylation

Normal Type II HBEC (15B), immortal Type II HBEC (M158V30),
and EL. clones were studied by Western blot. Positions of
phosphorylated and non-phosphorylated Rb are indicated in left.

60

 

 

E
92
g s. E
E S : ‘><" Z °'
01 o 3 CD 3, E g
5 3 r- C) 1- CD 1- g;
o N m < -‘ V -‘
p-RbL :
Rbr ..- -CII— — —.
.__¢....___ ______._4.:'_

Figure 27. Rb phosphorylation in normal and E. L. clones

Normal Type II HBEC (ISB), immortal Type II HBEC (M158V30), and
EL. clones were Studied by Western blot. positions of phosphorylated
and non-phosphorylated Rb are indicated in left.

E. P16 "3““

Recently, p16ink4a has been reported to play a role in the early event of
immortalization (Reznikoff et al., 1996; Noble et al., 1996). To understand whether p16
is involved in lifespan extension, the expression of p16 was examined. In the first
experiment studying HME20 E. L. clones (Fig. 28), there is detectable level of p16 in
normal Type 11 cells (20LB and 20RII). In these cells, p16 is localized in the cytoplasm.
The immortal M15SV30 cell line increased the level of p16 possibly because of
inactivated pr (Fig. 29). The E. L. clones derived from HME20 did not express p16. In
the second experiment, HME15 normal Type 11 cells were, unexpectedly, found not to
express p16 (Fig. 29). Among the extended lifespan clones studied (seven clones), only
XB7 expressed the p16. Another normal Type 11 cell culture (HME14) did express p16.
HME14 is one of four cell subjects that could not be successfully transformed. We
wondered if there might be a correlation between p16 expression and the ability to

acquire extended lifespan after X-ray irradiation. Therefore, the three normal Type II

61

cells derived from different women that were used for X-ray irradiation were assayed for

p16 expression. The results showed that HME5 and HME12 clearly express p16 (Fig. 30)

62

g

3%

'"19

30° 3%
a: ‘5 38
8N§§§8~§m<
ZNOONNOOOI<Q
”1),—Co G) 000
9020.996602ng
wfimofil‘jmom—I

l
!

P16

Figure 28. P16 expression and localization in E. L. clones derived
from HME20
Protein fraction indicated CYT: cytoplasmic ; NE: nuclear

 

x S 3 E
g o o
:3 nggrrggg
23§9wmr§ww<m
_ . -

Figure 29. P16 expression in normal and E. L. clones
Normal Type II HBEC ( M14B, M15B, M20LB), immortal Type II
HBEC(M158V30).

g E
:40!
OINW

P16 ...... ......

Figure 30. Expression of p16 in different normal Type II HBEC

63

Discussion

Based on substantial evidence that ionizing radiation is a breast carcinogen and the
observation that Type I HBEC is more susceptible to neOplastic transformation than Type
II HBEC (Kao et al., 1995), experiments were carried out to test if X-rays are effective
initiator of neoplastic transformation of a specific type of HBEC. Furthermore, by
characterizing the transformed cell clones with extended or infinite lifespan, an insight
into the genetic and molecular mechanisms for radiation -induced breast carcinogenesis
might be gained. The initial focus of the characterization is on certain genes known to
regulate the cell cycle and to maintain genome stability.

The major results of this study may be summarized as follows:

1. Clones with extended lifespan have been induced and isolated from Type II HBEC
after repeated low dose X-ray treatment. Twenty-two and two E. L. clones have been
isolated from HME15 and HME20, respectively. However, no E. L. clone was isolated
from four other different primary HBEC cultures.

2. These Type II E. L. clones failed to become immortal after prolonged culture or
with additional X-ray irradiation.

3. No B. L. clone was isolated from Type I HBEC with similar X-ray treatment.

4. The p53 and p21 proteins were frequently and concomitantly elevated in these
E. L. clones derived from Type II HBEC. However, these clones appear to contain
functional wild type p53, since they showed radiation-induced Gl arrest.

5. While the other Type II HBEC used in this study expressed the pl6/ink4a protein,

the HME15 which gave rise to many X-ray induced E.L. clones were unexpectedly

64

deficient in the expression of p16/ink4a. However, one of the E. L. clones derived from
this cell culture (XB7) re-expressed the p16 protein indicating that the p16 gene is
suppressed but not mutated. The experiment also confirmed that p16 may be up-regulated
in cells with inactivated Rb. The other E. L. clones examined showed non or weak
expression of p16.

6. While the parental Type II HBEC expressed the hyper-phosphorylated Rb,

the E. L. clones expressed both phosphorylated and unphosphorylated Rb.

These results have several implications and raise many new questions

a. Why do Type II E. L. clones fail to become immortal spontaneously or after

additional X-ray irradiation?

Most of the E. L. clones were isolated from HME15. Using the same cells, it was
found that both Type I and Type II cells were equally susceptible to transformation by
SV40 large T antigen to become E. L. clones. However, the Type II E. L. clones were not
easily immortalized (1/ 10) campared to Type I E. L. clones (10/ 1 1) after prolonged
growth (Chang, C. C. et al. unpublished results). Thus Type II HBEC are instn'nsically
not easily to become immortalized. Wazer et al. (1994) did report the isolation of a
immortal HBEC line after X-ray irradiation. The total dose used in their experiments (30
Gy) is higher than that used in this study. It is possible that, with continual repeated low
dose X-ray treatment of large population of cells, our E. L. clones will become immortal.

The frequency is, however, expected to be low as shown by Wazer et al. (1994).

65
b.What might be the reason for the inability for X-Rays to induce extended lifespan

in Type I HBEC?

The failure to obtain E. L. clones from Type I HBEC after X-ray irradiation may be
due to several reasons. First, the total number of Type I cells used in the experiments is
considerably less than Type II cells (about 1/4 - 1/5). Unlike SV40 large T antigen which
can inactivate p53 and Rb completely by complexing with the gene products, the X-rays
need to delete both alleles to transform if the target gene is a tumor suppressor gene. The
second possibility is that the protocol for X-ray treatment may not be right. We did
observe that Type I cells were more sensitive to radiation-induced inhibition of cell
proliferation. Perhaps a more frequent treatment with much lower dose each time would
be more effective. The third possibility is the difference between in vitro and in vivo. In
vivo, Type I HBEC receive signals by attaching to the extra-cellular matrix. While in
vitro, the cells were plated on plastic surface that sent different signals to the cells (
Trosko. JE, personal communication). In addition to that, Type I HBEC interact with
other types of cell in vivo may provide protection against radiation killing. Alternatively,
X-rays may function as an effective carcinogen at later stage but not at the initial stage.
There is evidence that X-rays are effective in converting immortal HBEC to weakly
tumorigenic cells (Kang et al., 1997)

c. Why were Type II HBEC derived from different women not equally

transforrnable by X-rays to acquire extended lifespan?

E.L. clones were isolated from only two of six different Type II HBEC treated with
X-ray. HME15 was the most transformable. The reason for the discrepancy in

transformability among different cultures is not clear. Since HME15 is the only one

66
among the five HBEC (HME5, 12, 14, 15 and 20) not expressing p16, it is tempting to

speculate that p16 deficiency may contribute to a cell culture’s susceptibility to be
transformed by X-rays.

d. Is there a role of elevated p53 in the expression of extended lifespan and in the

failure of E. L. clones to become immortal?

The E. L. Type II HBEC clones are frequently found to contain elevated level of p53
protein. The increased amount of p53 is the wild type form as indicated by the
concomitant increase of p21 and by the radiation-induced G1 arrest in these cells. Unless
there are different mechanisms to confer extended lifespan, the result of this studies with
X-rays are difficult to reconcile with the results of SV40 studies where the elimination of
p53 function leads to extended lifespan. The failure of Type II HBEC E. L. clone to
become immortal may not be due to the presence of elevated functional p53 since SV40
transformed Type II HBEC also were infrequently to become immortal. These cells
contain no functional p53 due to the presence of the SV40 large T antigen.

e. Why, unlike normal or immortal HBEC which express the phosphorylated form
(non-functional) of Rb, do the E. L. clones of Type II express both phosphorylated
and unphosphorylated form (functional) of Rb?

Consistent with what might be expected of elevated p53 and p21, the E. L. clones

were found to contain the unphosphorylated form of Rb in addition to the phosphorylated
form. This may reflect that most immortal and normal cells at the stage of analysis are

actively proliferating whereas E. L. clones contain considerable non-cycling cells.

67
f. Why does the level of p21 increase in the EL. clones?

Since p21 are associated with senescence, and deficiency in p21 in human fibroblast
lead to escape from senescence (Brown et al., 1997), it was expected that lower
expression of p21 might be found in E.L. clones in this study. The possible explanations
for the opposite result is that different types of cell may have different mechanisms to
enter senescence. In this case, p16 may play the major role in senescence in our study.
Another possibility is that both p21 and p16 are required for cells to enter senescence,
inactivation of either one could result in bypass of senescence. The presence of elevated
levels of p53 and p21 in E. L. clones may indicate that these cells contain both
proliferating and senescent cells.

The results of this study have several implications. First, suppose the failure to
transform Type I HBEC by X-rays is due to the use of small population of cells and the
use of improper protocol, new effort to obtain E. L. and immortal Type I HBEC should
use a different protocol taking these factors and other variable into consideration. for
example, organoids formed by Type I cells on Matrigel may be used for neoplastic
transformation by X-rays. Second, the failure of Type II E. L. clones to become immortal
is consistent with the results form SV40 studies, indicating that Type II HBEC may not
be consider as target cells for neoplastic transformation. Third, the deficient of p16 found
in the most transforrnable culture (HME15) suggests that p16 may not be expressed in all
normal HBEC and that this deficiency may be correlated with susceptibility to
environmental agents induced neoplastic transformation. This should be tested in future

studies

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68

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