Differential Expression of Multiple MDM2 Messenger …clincancerres.aacrjournals.org/content/clincanres/1/1/71.full.pdfDifferential Expression of Multiple MDM2 Messenger RNAs and Proteins
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Vol. 1, 71-80, Jantiar�’ 1995 Clinical Cancer Research 71
Differential Expression of Multiple MDM2 Messenger RNAs and
Proteins in Normal and Tumorigenic Breast Epithelial Cells
Jean M. Gudas,’ Hoang Nguyen,2
Regina C. Klein, Dai Katayose, Prem Seth,
and Kenneth H. Cowan
Medicine Branch, Division of Cancer Treatment, Medical Breast
Cancer Section, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT
The MDM2 gene is a nuclear phosphoprotein that is
regulated by p53 and functions, in one capacity, to inhibit
the transcriptional activity of the wild-type p53 protein.
Multiple MDM2 transcripts were detected in human breast
epithelial cells. In estrogen receptor-negative normal, im-
mortal, and tumorigenic breast epithelial cells, we found agood correlation between MDM2 mRNA levels and expres-
sion of wild-type p53. When wild-type p53 was overex-
pressed in estrogen receptor-negative tumor cells containing
a mutant or no endogenous p53, MDM2 mRNA levels in-creased significantly, indicating that wild-type p53 positively
influences MDM2 mRNA levels in these tumor cells. Because
all estrogen receptor-positive breast tumor cells had high
MDM2 mRNA levels regardless of the status of their endo-genous p53 protein, other factors likely influence MDM2
expression in these cells Distinct MDM2 proteins (range, Mr
54,000-68,000 and 90,000-100,000, respectively) were dif-
ferentially expressed in human breast epithelial cells. The
lower molecular weight MDM2 proteins were most abun-
dant in the normal mammary cells but present at varyinglevels in many of the tumor cells examined. MDM2 was a
nuclear protein; however, nuclear staining intensity did notalways correlate with the amount of MDM2-immunoreac-
tive protein as determined by Western blot analysis. This
discrepancy suggests that MDM2 interacts with novel cellu-
lar proteins in different kinds of breast epithelial cells.
INTRODUCTION
The human homologue of the mouse MDM2 gene encodes
a zinc finger-containing protein that is a putative transcription
factor (1, 2). Overexpression of this gene in BALB/c 3T3 cells
increases their tumonigenic potential, suggesting that the MDM2
protein possesses oncogenic activity. Further evidence that
MDM2 plays a role in the process of cell transformation derives
from experiments where this protein was shown to complex
with and inhibit the transcriptional activity of wild-type p53 (3,
Received 8/16/94; accepted 10/20/94.
I To whom requests for reprints should be addressed, at MedicineBranch, Division of Cancer Treatment, National Cancer Institute, Build-ing 10, Room 12N226, Bethesda, MD 20892.
2 Supported by a Cooperative Research Development Agreement pro-vided by the Centocor Corp., Malvern, PA.
4). MDM2 interacts with the amino terminal acidic activation
domain of the p53 protein, raising the possibility that MDM2
inhibits p53 function by disrupting its interaction with the gen-
enal transcription machinery (5).
Recent evidence indicates that MDM2 is one of the down-
stream effector genes in cells that express a wild-type p53
protein (6, 7). The first intron of the MDM2 gene contains a p53
DNA binding site. This element confers wild-type p53 respon-
sivity to the endogenous MDM2 gene and to reporter genes
when placed upstream in a hetenologous promoter (7). More
recent data suggest that this p53 response element may actually
be part of an internal, cryptic p53-dependent promoter (8).
When considered together, these observations suggest that neg-
ulation of p53 and MDM2 are intimately linked and that these
two proteins may act in concert to control cell proliferation.
Our laboratory has been interested in regulatory mecha-
nisms that play a role in the genesis of breast cancer. The
importance of p53 in this process is underscored by the finding
that -40% of primary human breast tumors harbor mutations in
the coding sequences of the p53 gene (9, 10). Moreover, recent
studies indicate that expression of mutant p53 is an important
prognostic marker for node-negative breast cancers (1 1, 12).
Presumably, the remaining tumors utilize other mechanisms to
bypass the negative regulatory effects of the wild-type p.53 gene.
One possibility is that these tumor cells can up-regulate the
expression of MDM2 to a level sufficient for binding and
titrating the effects of wild-type p53. Such a mechanism has
been proposed to explain the ovenexpression of MDM2 in soft
tissue sarcomas, osteosancomas, and a subset of malignant gli-
omas (13-17). Recently, Chen et a!. (18) reported that the
amplification of MDM2 in osteosancoma cells resulted in inhi-
bition of wild-type p53-induced growth arrest following treat-
ment with ionizing radiation. These results have provided im-
portant evidence demonstrating that MDM2 overexpression can
affect p53 function in a known physiological pathway. To date,
amplification and overexpression of MDM2 has not been shown
to play a significant role in the genesis of human epithelial
tumors (19-21).
To begin to understand the complex regulation between
these two cellular proteins, we examined levels of p53 and
MDM2 mRNA and protein in human breast epithelial cells. We
determined the sizes of the mRNAs and proteins for MDM2
present in mammary epithelial cells at different stages of cell
transformation. One goal of oun study was to determine whether
a relationship existed between p53 levels and corresponding
levels of MDM2 in mammary epithelial cells that expressed
wild-type versus mutant p53 proteins. Because MDM2 levels
were high in ER�3 breast cancer cells regardless of their p53
3 The abbreviations used are: ER, estrogen receptor; NMECs, normalhuman mammary epithelial cells.
Fig. I Northern blot analysis of MDM2 and p53 RNA in human breast epithelial cells. The p53 status of the cells was obtained from several sources
and, if known, is indicated in parentheses (30, 31, 33, 36). RNA was isolated from three independently derived normal human mammary epithelialcells HMEC 1-3 (Wi’), immortalized, nontumonigenic HBL100, MCF-10 (Wi’) and 184B5 (Wi’) cells, estrogen receptor-positive MCF-7 (WT),ZR7S-1 (WT), T47-D (codon 194 mutation) and BT-474 (codon 285 mutation) tumor cells and estrogen receptor-negative MDA-MB-157 (null),
cancer cells, electrophonesed, transferred, and hybridized with probes for the MDM2, p53. and 36B4 genes, respectively. The lane designated SJSA-1contains RNA from an osteosarcoma line that has an amplification of the MDM2 gene. Arrows to the left of the top panel, sizes of the MDM2transcripts present in these cells. Single panel to the right, a shorter exposure of the lane containing RNA from the SJSA-1 cell line.
MDM2 in normal and immortal versus transformed human
mammary epithelial cells. For these experiments total RNA was
isolated from exponentially growing populations of NMECs
derived from reduction mammoplasties, immortalized but non-
tumonigenic mammary epithelial cells and estrogen receptor-
positive and -negative breast cancer cell lines. Normal and
immortalized, nontumonigenic breast cells grown in culture do
not express the estrogen receptor. This observation likely re-
fleets the biology of mammary epithelial cells as very few cells
are ER� in normal nonlactating mammary glands (27, 28). To
ascertain the estrogen receptor status of the various cells, we
probed the filter for ER mRNA (Fig.i, Panel 2). As expected,
only the ER� breast tumor cells had detectable levels of estro-
gen receptor mRNA.
Relatively high levels of MDM2 mRNA were present in all
three independently derived ER, NMEC strains (Lanes 1-3).
The amount of MDM2 mRNA detected in spontaneously arising
MCF1O (Lane 5) and carcinogen-induced i84B5 (Lane 6) ER,
immortalized, nontumonigenic cells was similar to the amount
present in NMECs. In contrast, HBL100 mammary epithelial
cells, that were immortalized by SV4O large T antigen, ex-
pressed a much lower level of MDM2 mRNA (Lane 4). This is
an important observation as SV4O T antigen binds to and mac-
tivates the function of wild-type p53. The four estrogen receptor
positive breast cancer cell lines MCF-7, ZR7S-1, T47-D and
BT474 (Lanes 7-10) expressed similar MDM2 mRNA levels to
those found in normal mammary epithelial cells. In contrast, the
five estrogen receptor negative breast cancer cell lines exam-
ined, MDA-MB-157, MDA-MB-231, MDA-MB-468, SKBr-3,
and HSS78T, all expressed MDM2 RNA levels that were S to iO
fold lower than in any of the other cells analyzed (Lanes Il-is).
Although all human mammary epithelial cells expressed readily
detectable MDM2 transcripts, the abundance of this mRNA was
well below the amount present in the overexpressing human
osteosarcoma cell line, SJSA-1 (16).
MDM2 mRNAs having several different molecular
weights were detected in our panel of mammary epithelial cells.
To determine the sizes of the various transcripts, identical
Fig. 3 Western blot analysis of MDM2 and p53 proteins in human breast epithelial cells. Total protein extracts from exponentially growing normal
mammary epithelial cells (Lanes 1 and 2), 184B5 cells (Lane 3), immortalized MCF-l0 (Lane 4), estrogen receptor-positive MCF-7 (Lane 5), T47-D(Lane 6), ZR7S-l (Lane 7), and BT-474 (Lane 8) tumor cells, and estrogen receptor-negative SKBr3 (Lane 9), HS578T (Lane 10), MDA-MB-157(Lane 11), MDA-MB-231 (Lane 12), and MDA-MB-468 (Lane 13) tumor cells were prepared in Laemmli lysis buffer. Protein (50 �xWlane) was
separated in an 8% SDS polyacylamide gel, electroblotted onto nitrocellulose, and the membranes were reacted with antibodies corresponding to
MDM2, PS3, or actin. Protein molecular weight markers that were coelectrophoresed and transferred are indicated on the left of each panel. Lanedesignated SJSA-l contains protein extract from osteosarcoma cells that ovenexpress the MDM2 protein.
p53 in the cells (6, 7). To determine whether there was a
correlation between MDM2 and p53 protein levels in different
mammary epithelial cells, we performed p53 Western blot anal-
yses on the same extracts. These results showed no apparent
correlation between the absolute levels of p53 and MDM2
proteins in these cells.
A more critical consideration in these comparisons is
whether the p53 expressed in the various cells is mutant or wild
type. Normal and immortalized, nontumonigenic breast cells
grown in culture do not express the estrogen receptor (Fig. 1).
This finding likely reflects the biology of mammary epithelial
cells since very few cells are ER� in normal nonlactating
mammary glands (27, 28). The normal and immortal 184B5
mammary epithelial cells used in these studies express a wild-
type p53 protein that has increased stability (30-32). The p.53
gene in the immortalized MCF1O cells has been sequenced
along its entirety and shown to be wild type.5 All of these cells
expressed relatively high levels of MDM2 protein. In contrast,
the ER tumor cells (Lanes 10-14) expressed either a mutant on
no p53 and had relatively low levels of MDM2 mRNA and
protein (33). Thus, when examining ER mammary epithelial
cells, we found a good correlation between wild-type p53 ex-
pression and corresponding levels of MDM2 mRNA and the Mr
90,000 MDM2 protein.
The same positive correlation between wild-type p53 and
MDM2 protein expression did not hold true for the ER� breast
cancer cells. All of the ER� breast cancer cells expressed
relatively high levels of full length MDM2 protein when com-
pared to the ER cells. MCF-7 and ZR75-l cells express a
wild-type p53 protein (34, 35); however, T47-D and BT-474
cells both express mutant p53 (33, 36). Thus, factors other than
p53 are likely responsible for the high levels of MDM2 mRNA
and protein in ER� breast tumor cells. Among the various cell
tein using Western blot analysis (Fig. 3); however, considerable
variation in immunocytochemical staining patterns was ob-
served. The NMECs and immortalized l84B5 mammary epi-
thelial cells showed distinct nuclear staining for MDM2 with
some variation in intensity from one cell to another (Fig. 6A).
When the same cells were stained for p53 using the PAb 1801
antibody, a similar pattern of heterogenous nuclear staining was
observed. In contrast another immortalized, mammary epithelial
cell line MCF-10 showed less intense and more variable staining
for both MDM2 and p53 proteins. The decreased staining in-
tensity observed in these cells may be due to the slightly lower
levels of protein accumulation as determined by Western blot-
ting (Fig. 3).
The estrogen receptor-positive breast epithelial lines all
contained relatively high levels of MDM2 protein when exam-
med using Western blots (Fig. 3). However, when analyzed
immunocytochemically, the ER� tumor cells stained poorly, if
at all, for MDM2. In MCF-7 cells the small amount of peninu-
clear staining obtained with the MDM2 antibody is likely non-
specific because we see similar results using isotypic monoclo-
nal antibodies for other cellular proteins that are not expressed
36B4
Fig. 4 A, infection of ER� tumor cells with an adenovirus vector
containing a wild-type gene. Protein lysate prepared from exponentially
growing MDA-MB-157 and MDA-MB-231 ER tumor cells (Lanes 1
and 4), cells infected with a control adenovirus vector (Lanes 2 and 5),
and cells infected with AdWTpS3 virus (Lanes 3 and 6) was examined
for p53 expression using Western blot analysis. B. RNA was isolatedfrom cells treated as described in the legend to A and examined forMDM2 expression using Northern blot analysis. Lower panel, 36B4
expression from the same filter.
infected with a control virus lacking the p53 gene (Fig. 4A,
Lanes 2 and 5). In contrast, cells infected with a virus carrying
the wild-type p53 gene expressed significantly increased levels
of p53 protein (Fig. 4A, Lanes 3 and 6).
MDM2 mRNA levels were examined from cells indepen-
dently infected with the same viral constructs (Fig. 4B). MDM2
mRNA levels were relatively low in the uninfected cells (Fig.
4B, Lanes I and 4) and in the cells infected with a control virus
(Fig. 4B, Lanes 2 and 5). MDA-MB-157 and MDA-MB-231
cells expressing a wild-type p53 protein showed 6- and 10-fold
respective increases in MDM2 mRNA levels (Fig. 4B, Lanes 3
and 6). These increases in MDM2 mRNA levels are in accon-
dance with the finding of slightly higher levels of wild-type p53
protein in the MDA-MB-231 cells. When considered together
these data support the idea that wild-type p53 positively influ-
Fig. 5 Northern blot analysis of MDM2 mRNA in ER� cells following estrogen stimulation. MCF-7 and ZR75-l cells were depleted of estrogen
for S days and subsequently refed with medium containing charcoal-stripped serum and 10 nM �3-estradiol. At the times indicated above each lanetotal RNA was prepared, electrophoretically separated, transferred to filters, and probed for expression of MDM2 (upper panels), p52 (middle panels),
and 36B4 (lower panels). Large and small arrows, migration of the 6.7-kilobase and 4.5-kilobase MDM2 transcripts. respectively.
in these cells.6 Very few nuclei from ER� ZR7S-1 cells stained
for MDM2 protein and some cells may show some cytoplasmic
localization. Interestingly, staining of the ZR75-i cells for p53
revealed a distinct cytoplasmic staining that was highly local-
ized within discrete foci. To our knowledge, a similar staining
pattern for p53 has not been described in other cell types.
The ER, MDA-MB-231 breast cancer cells had very low
levels of MDM2 when examined usingWestern blot analysis
(Fig. 3) and did not stain at all for MDM2. Two other ER
breast cancer cell lines that expressed very low levels of MDM2
protein by immunoprecipitation and Western blotting, MDA-
MB-157 and MDA-MB-453, were also negative for MDM2
when examined immunocytochemically (data not shown). We
believe that the level of MDM2 protein may be below the
detection limit by immunocytochemistry in the ER breast
cancer cells. As expected for cells that express a mutant p53, all
nuclei in MDA-MB-231 cells stained intensely for p53. As
controls for the immunocytochemistry, we show that SJSA-1
cells, which overexpress MDM2, stained intensely for this an-
tigen while very few nuclei from these cells were positive for
p53 (Fig. 6).
DISCUSSION
In this study we report two independent observations con-
cerning MDM2 expression in human breast epithelial cells. The
first finding is that a good correlation exists between expression
of wild-type p53 in normal and immortalized mammary epithe-
hal cells with accumulation of relatively high levels of MDM2
mRNA and protein in the same cells. Because the magnitude of
the difference in relative MDM2 mRNA levels was greaten than
that observed for corresponding MDM2 protein levels, other
factors, including protein stability and translational efficiency,
likely play a role in determining the total amount of MDM2
protein in a cell. The normal and immortalized human mammary
epithelial cells used for these studies are estrogen receptor
negative and express a wild-type p53 protein. The wild-type p53
is unusual in these cells because it has a half-life of -3 h (30r
compared to a half-life of -20 mm in many other cell types (30,
39, 40).
In contrast, all of the estrogen receptor-negative breast
tumor cell lines studied had either a mutant on no p53 protein
and expressed significantly less MDM2 mRNA and protein.
Because the ER cell lines we examined all had mutations in the
conserved DNA binding domain of the protein (41), they are all
likely defective in their ability to transactivate cellular target
genes. Previous studies from other laboratories have demon-
strated a consensus p53 DNA binding site in the MDM2 gene
which is necessary for regulation by wild-type p53 (6, 7). To
Several different MDM2 transcripts, ranging in size from
4.5 to 12.5 kilobases, were detected in mammary epithelial cells.
At present we do not know if these various mRNA species have
functional significance or if some of the higher molecular
weight transcripts represent partially on alternatively processed
MDM2 mRNAs. Interestingly, evidence from DNA sequencing
studies indicates that both the human and mouse MDM2
mRNAs can be alternatively spliced (1, 49). Because all mRNA
bands hybridized with probes obtained from both the 5’ and 3’
end of the coding region of the human MDM2 eDNA, we
believe that they are all bona fide MDM2 transcripts.
Results from immunoprecipitation and Western blot anal-
yses showed a complex pattern of many different MDM2 pro-
teins in human breast epithelial cells. The predominant protein
detected from all mammary epithelial cells migrated as a Mr
90,0000 band. This form likely represents the full-length
MDM2 protein while the smaller, -57,000 form, may be an
alternatively spliced variant as described in mouse and rat cells
(I, 29). Haines et al. (29) have necently reported that the
different molecular weights of the MDM2 proteins in mouse
cells represent alternatively spliced variants. Moreover, they
have demonstrated that these proteins differ in their ability to
physically associate with, and thus inhibit, the function of wild-
type p53 (29). When considered along with the finding of
multiple mRNA species, we believe that the different forms
of MDM2 present in breast epithelial cells likely represent
distinct MDM2 proteins that may have both overlapping and
unique biological functions.
Immunocytochemical staining for MDM2 showed that the
protein was nuclear in breast epithelial cells; however, the
intensity and number of positive cells did not always correlate
with the level of protein detectable by biochemical methods.
Some of the variation in nuclear MDM2 staining seen in the
NMECs and l84B5 cells may reflect differences in cell cycle
distribution as we have previously shown that these cells stain
variably for p53, depending upon their position in the cell cycle
(32). ER� MCF-7, ZR75-1, and BT474 (data not shown) cells
showed little or no staining for MDM2 although they had
comparable amounts of the Mr 90,000 MDM2 when assayed
using Western blot analysis (Fig. 3). One possibile explanation
for the anomolous immunocytochemical results in ER� cells is
that MDM2 is somehow sequestered in a larger complex that is
only dissociated on lysis of the cells. We are presently perform-
ing more detailed experiments to test this hypothesis. With the
exception of a unique cytoplasmic focal staining pattern for p53
observed in ZR7S-1 cells, our results for p53 staining in breast
epithelial cells are similar to those reported by other investiga-
tons (30, 33, 50).
Increasing evidence suggests that p53 and its downstream
effector proteins MDM2 and CIP/Waf-1 play a role in deter-
mining cellular responses to endogenous and exogenous signals.
The differential expression of multiple MDM2 mRNAs and
proteins raises the possibility that these products may perform
distinct functions in mammary epithelial cells at different stages
of tumor development. We believe that further study of p53 and
its downstream effector proteins MDM2 and CIP-1 (51, 52)
should enhance our understanding of the processes of cell trans-
formation and the means by which cells modulate their response
to DNA damage and environmental stress. A better understand-
ing of these cellular processes should in turn translate into more
specific, and, hopefully, more effective therapeutic approaches
for the treatment of breast cancers.
ACKNOWLEDGMENTS
We thank Drs. Arnold Levine and Vincent Marachel for providing
us with the 5BlO monoclonal antibody. We are also grateful to Dr. Bert
Vogelstein for providing the human MDM2 eDNA clone, Dr. Thomas
Look for the SJSA-1 osteosarcoma cells, and Dr. Erasmus Schneider for
helpful comments on the manuscript.
REFERENCES
1. Olson, D. C., Marechal, V., Momand, J., Chen, J., Romocki, C., andLevine, A. J. Identification and characterization of multiple mdm-2proteins and mdm-2-p53 protein complexes. Oneogene, 8: 2353-2360,
1993.
2. Momand, J., Zambetti, G. P., Olson, D. C., George, D., and Levine,A. J. The mdm-2 oneogene product forms a complex with the p53protein and inhibits p53-mediated transactivation. Cell, 69: 1237-1245,
1992.
3. Oliner, J. D., Pietenpol, J. A., Thiagalingam, S., Gyunis, J., Kinzler,K. W., and Vogelstein, B. Oncoprotein MDM2 conceals the activationdomain of tumour suppressor p53. Nature (Land.), 362: 857-860, 1993.
4. Finlay, C. A. The ,ndm-2 oncogene can overcome wild-type p53suppression of transformed cell growth. Mol. Cell. Biol., /3: 301-306,
1993.
5. Chen, J., Marechal, V., and Levine, A. J. Mapping of the p53 andmdm-2 interaction domains. Mol. Cell. Biol., 13: 4107-41 14, 1993.
6. Barak, Y., Juven, T., Haffner, R., and Oren, M. mdm-2 expression isinduced by wild type p53. EMBO J., 12: 461-468, 1993.
7. Wu, X., Bayle, J. H., Olson, D., and Levine, A. J. The p53-mdm-2autoregulatory feedback loop. Genes & Dev., 7: 1126-1 132, 1993.
8. Juven, T., Barak, Y., Zauberman, A., George, D. L., and Oren, M.
Wild type p53 can mediate sequence-specific transactivation of aninternal promoter within the rndm2 gene. Oncogene, 8: 341 1-34 16,
1993.
9. Davidoff, A. M., Humphrey, P. A., Iglehart, J. D.. and Marks, J. R.Genetic basis for p53 overexpression in human breast cancer. Proc. Nati.Acad. Sci. USA, 88: 5006-5010, 1991.
10. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53Mutations in human cancers. Science (Washington DC), 253: 49-53,
1991.
11. AImed, D. C., Clark, G. M., Elledge. R., Fuqua, S. A. W., Brown,R. W., Chamness, G. C., Osborne, C. K., and McGuire, W. L. Associ-ation of p53 expression with tumor cell proliferation rate and clinicaloutcome in node-negative breast cancer. J. NatI. Cancer Inst., 85:
200-206, 1993.
12. Barnes, D. M., Dublin, E. A., Fisher, C. J., Levison, D. A., and
Millis, R. R. Immunohistochemical detection of p53 protein in mam-
mary carcinoma: an important new indicator of prognosis? Hum.
Pathol., 24: 469-76, 1993.
13. Oliner, J. D., Kinzler, K. W., Meltzen, P. S., George, D. L., andVogelstein, B. Amplification of a gene encoding a p53-associated pro-
tein in human sarcomas. Nature (Land.), 358: 80-83, 1992.
14. Leach, F. S., Tokino, T., Meltzer, P., Burrell, M., Oliner, J. D.,Smith, S., Hill, D. E., Sidransky, D., Kinzler, K. W., and Vogeistein, B.p53 mutation and MDM2 amplification in human soft tissue sarcomas.
Cancer Res., 53: 2231-2234, 1993.
15. Ladanyi, M., Cha, C., Lewis, R., Jhanwar, S. C., Huvos, A. G., and
Healey, J. H. MDM2 gene amplification in metastatic osteosarcoma.Cancer Res., 53: 16-18, 1993.
16. Khatib, Z. A., Matsushime, H., Valentine, M., Shapiro, D. N., Shern,C. J., and Look, A. T. Coamplification of the CDK4 gene with MDM2and GLI in human osteosarcomas. Cancer Res., 53: 5535-5541, 1993.
17. Reifenberger, G., Liu, L., Ichimura, K., Schmidt, E. E., and Collins,V. P. Amplification and ovenexpression of the MDM2 gene in a subsetof human malignant glioblastomas without p53 mutations. Cancer Res.,53: 2736-2739, 1993.
18. Chen, C.-W., Oliner, J. D., Zhan, Q., Fornace, A. J. J., Vogelstein,B., and Kastan, M. Interactions between p53 and MDM2 in a mamma-han cell cycle checkpoint pathway. Proc. Natl. Acad. Sci. USA, 91:
2684-2688, 1994.
19. Waber, P. G., and Nisen, P. D. Infrequency of MDM2 amplificationin pediatric solid tumors and lack of association with p53 mutations in
adult squamous cell carcinomas. Cancer Res., 53: 6028-6030, 1993.
20. Kessis, T. D., Slebos, R. J., Han, S. M., Shah, K., Bosch, X. F.,Munoz, N., Hednick, L., and Cho, K. R. p53 mutations and MDM2amplifications are uncommon in primary carcinomas of the uterine
cervix. Am. J. Pathol., 143: 1398-1405, 1993.
21. Esteve, A., Lehman, T., Jiang, W., Weinstein, I. B., Harris, C. C.,Ruol, A., Peracehia, A., Montesano, R., and Hollstein, M. Correlation ofp53 mutations with epidermal growth factor overexpnession and absence
of mdm2 amplification in human esophageal carcinomas. Mol. Car-
cinog., 8: 306-311, 1993.
22. Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson, W. D. J.,Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F., andBrooks, S. C. Isolation and characterization of a spontaneously immor-talized human breast epithelial cell line, MCF-10. Cancer Res., 50:
6075-6086, 1990.
23. Graham, K. A., Trent, J. M., Osborne, C. K., McGrath, C. M.,Minden, M. D., and Buick, R. N. The use of restriction fragment
polymorphisms to identify the cell line MCF-7. Breast Cancer Treat.
Res., 8: 29-34, 1986.
24. Stampfer, M. R., and Bartley, J. C. Human mammary epithelial
cells in culture: differentiation and transformation. In: Lippman, M. E.and Dickson, R. B. (eds.), Breast Cancer: Cellular and MolecularBiology, pp. 1-23. Boston: Kluwer Academic Publishers, 1988.
25. Gudas, J. M., Knight, G. B., and Pardee, A. B. Nuclear posttran-
scniptional processing of thymidine kinase mRNA at the onset of DNAsynthesis. Proc. NatI. Acad. Sci. USA, 85: 4705-4709, 1988.
26. Masiakowski, P., Bneathnach, R., Bloch, J., Gannon, F., Krust, A.,and Chambon, P. Cloning of eDNA sequences of hormone-regulatedgenes from the MCF-7 human breast cancer cell line. Nucleic Acids
Res., 10: 7895-7903, 1982.
27. Peterson, 0. W., Hoyer, P. E., and Van Deurs, B. Frequency and
distribution of estrogen receptor positive cells in normal, non-lactating
human breast tissue. Cancer Res., 47: 5748-5751, 1987.
28. Ricketts, D., Turnbull, L., Ryall, G., Bakhshi, R., Rawson, N. S. B.,Gazet, J.-C., Nolan, C., and Coombes, R. C. Estrogen and progesteronereceptors in the normal female breast. Cancer Res., 51: 1817-1822,
1991.
29. Haines, D. S., Landers, J. E., Engle, L. J., and George, D. L.Physical and functional interaction between wild-type p53 and mdm2
proteins. Mol. Cell. Biol., 14: 1171-1178, 1994.
30. Delmolino, L., Band, H., and Band, V. Expression and stability of
p53 protein in normal human mammary epithelial cells. Carcinogenesis,
14: 827-832, 1993.
31. Lehman, T. A., Modali, R., Boukamp, P., Stanek, J., Bennett, W. P.,
Welsh, J. A., Metcalf, R. A., Stampfer, M. R., Fusenig, N., Rogan, E.M., and Harris, C. C. p53 mutations in human immortalized epithelialcells. Cancinogenesis, 14: 833-839, 1993.
32. Gudas, J. M., Oka, M., Diella, F., Trepel, J., and Cowan, K. H.Expression of wild-type p53 during the cell cycle in normal humanmammary epithelial cells. Cell Growth & Differ., 5: 295-304, 1994.
33. Bartek, J., Iggo, R., Gannon, J., and Lane, D. P. Genetic and
immunochemical analysis of mutant p53 in human breast cancer cell
lines. Oncogene, 5: 893-899, 1990.
34. Casey, G., Lo-Hsueh, M., Lopez, M. E., Vogelstein, B., and Stan-
bridge, E. J. Growth suppression of human breast cancer cells by the
introduction of a wild-type p53 gene. Oncogene, 6: 1791-1797, 1991.
35. Takahashi, K., Sumimoto, H., Suzuki, K., and Ono, T. Protein
synthesis-dependent cytoplasmic translocation of p53 protein after se-
rum stimulation of growth-arrested MCF-7 cells. Mol. Cancinog., 8:
58-66, 1993.
36. Haldan, S., Negnini, M., Monne, M., Sabbioni, S., and Croce, C. M.Down-regulation of bcl-2 by p53 in breast cancer cells. Cancer Res., 54:
2095-2097, 1994.
37. Berthois, Y., Katzenellenbogen, J. A., and Katzenellenbogen, B. S.Phenol red in tissue culture media is a weak estrogen: implicationsconcerning the study of estrogen-responsive cells in culture. Proc. NatI.
Acad. Sci. USA, 83: 2496-2500, 1986.
38. Darbne, P. D. Steroids and steroid receptors in growth control of
cultured breast cancer cells. Int. J. Cancer, 5(Suppl.): 67-75, 1990.
39. Gronostajski, R., Goldberg, A. L., and Pardee, A. B. Energy ne-quinement for degradation of tumor-associated protein p53. Mol. Cell.
Biol., 4: 442-448, 1984.
40. Mora, P. T., Chandrasekaran, K., Hoffman, J. C., and McFarland,V. W. Quantitation of a 55K cellular protein: similar amount andinstability in normal and malignant mouse cells. Mol. Cell. Biol., 2:
763-771, 1982.
41. Cho, Y., Gonna, S., Jeffrey, P. D., and Pavletich, N. P. Crystal
structure of a p53 tumor suppressor-DNA complex: understanding tu-
42. Blanco, G., Alavaikko, M., Ojala, A., Collan, Y. Heikkinen, M.,
Hietanen, T., Aine, R., and Taskinen, P. J. Estrogen and progesteronereceptors in breast cancer: relationships to tumor histopathology and
survival of patients. Anticancer Res., 4: 383, 1984.
43. Singh, L., Wilson, A. J., Baum, M., Whimsten, W. F., Birch, I. H.,Jackson, I. M. Lowney, C., and Palmer, M. K. The relationship betweenhistological grade, oestnogen receptor status, events and survival at 8
years in the NATO (Nolvadex) trial. Br. J. Cancer, 57: 612, 1988.
44. Moscow, J. A., Townsend, A. J., and Goldsmith, M. E. Isolation of
the human anioic glutathione 5-transferase eDNA and the relation of itsgene expression to estrogen-receptor content in primary breast cancer.Proc. NatI. Acad. Sci. USA, 85: 6518- 6522, 1988.
45. Morrow, C. S., Chiu, J., and Cowan, K. H. Posttranscriptional
control of glutathione-S-tnansferase pi gene expression in human breastcancer cells. J. Biol. Chem., 267: 10544-10550, 1992.
46. Townsend, A. J., Morrow, C. S., and Sinha, B. Selenium-dependentglutathione peroxidase expression is inversely related to estrogen recep-ton content of human breast cancer cells. Cancer Commun., 3: 265-270,
1991.
47. Saeed, M., Shao, Z-M., Hussain, A., and Fontana, J. A. The p53-
binding protein MDM2 gene is differentially expressed in human breast
carcinoma. Cancer Res., 53: 3226-3228, 1993.
48. Jiang, S-Y., and Jordan, V. C. Growth regulation of estrogen
receptor-negative breast cancer cells transfected with complementaryDNAs for estrogen receptor. J. Natl. Cancer Inst., 84: 580-591, 1992.
49. Fakhanzadeh, S. S., Trusko, S. P., and George, D. L. Tumonigenicpotential associated with enhanced expression of a gene that is amplified
in a mouse tumor cell line. EMBO J., 10: 1565-1569, 1991.
50. Banouch, D. A., Xu, M., Tewani, A., Chnistman, J. K., and Lane, M.
A. In situ dephosphorylation of p53 by calf intestinal alkaline phos-
phatase treatment. Oncogene, 7: 2351-2353, 1992.
51. Harper, J. W., Adami, G. R., Wei, N., Keyomansi, K., and Elledge,S. J. The p21 Cdk-interacting protein Cipi is a potent inhibitor ofGi-cyclin dependent kinases. Cell, 75: 805-816, 1993.
52. El-Diery, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Par-sons, R., Trent, J. M., Lin, D., Mencer, W. E., Kinzler, K. W., andVogelstein, B. WAF1, a potential mediator of p53 tumor suppression.Cell, 75: 817-825, 1993.
1995;1:71-80. Clin Cancer Res J M Gudas, H Nguyen, R C Klein, et al. proteins in normal and tumorigenic breast epithelial cells.Differential expression of multiple MDM2 messenger RNAs and