Progressive increase of glucose transporter-3 (GLUT-3) expression in estrogen-induced breast carcinogenesis.

INTRODUCTION: Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. A family of glucose transporters (GLUTs) facilitates glucose movement across the plasma membranes in a tumor-specific manner. Glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12, have been previously shown in breast cancer cells and are found to be associated with poor prognosis. In addition, it has been shown that estrogen plays critical roles in GLUT regulation, however, the stage-specific GLUT regulation of mammary carcinogenesis is unclear. METHODS: GLUT expression patterns were investigated in an in vitro-in vivo progressive, estrogen-induced, mammary carcinogenesis model which consisted of four cell lines, with same genetic background. In this model, different stages of tumor initiation and progression are represented, MCF-10F being the normal stage, E2 cells the transformed stage by estrogen, C5 cells, the invasive stage, and T4 cells the tumorigenic stage. In addition, loss of ductulogenesis and solid mass formation in collagen matrix and invasiveness of the cells were counted. RESULTS: Real time PCR showed that GLUT1 expression was downregulated in MCF10F after treatment with 17ß-estradiol (E2), and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasive capacity, whereas, GLUT-12 was downregulated in C5 and T4 cells. CONCLUSION: Estrogen-induced malignant transformation is associated with remarkable and progressive GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation.

Breast cancer prevention.

We have developed a new approach for breast cancer prevention, capitalizing in the preventive effect of early first full-term pregnancy, hormonally induced differentiation and our ability to identify specific genomic signatures that allow us to predict risk reduction. Early pregnancy imprints in the breast permanent genomic changes or a 'signature' that reduces the susceptibility of this organ to cancer. At cellular level, what we have achieved is the shifting of the Stem Cell 1 population, highly susceptible to cancer, to a population of Stem Cell 2 that is refractory to carcinogenesis. In a case-control study, we have compared the gene expression profile in normal breast tissue from nulliparous and parous postmenopausal women with (case) and without (control) breast cancer. We have determined that early first full-term pregnancy induces a specific genomic signature in the postmenopausal breast that is the biomarker for the Stem cell 2. The Stem cell 2 contains specific genes controlling transcription, RNA processing, immune response, apoptosis and DNA repair. We have further detected in the plasma, using an ELISA assay, the proteins coded by the gene signature. We are developing clinical trials to demonstrate the proof of the principle that r-hCG can induce in the human breast a genomic signature of the Stem cell 2. This is a concept that challenges the currently available chemopreventive agents that need to be given for extended periods for maintaining the suppression of a specific metabolic pathway or the abrogation of the function of an organ.

Immune-surveillance and programmed cell death-related genes are significantly overexpressed in the normal breast epithelium of postmenopausal parous women.

Endocrine and reproductive influences significantly affect the lifetime risk of breast cancer. Nulliparity is one of the most firmly established risk factors for breast cancer, whereas early full-term pregnancy and parity confer a significant protection. The breast attains its maximum development during pregnancy and lactation. After menopause the breast regresses in both nulliparous and parous women containing lobular structures designated lobules type 1 (Lob 1). We have postulated that the degree of differentiation acquired through early pregnancy changes the 'genomic signature' that differentiates the Lob 1 from the early parous women from that of the nulliparous women by shifting the Stem cell 1 to a Stem cell 2, making this the mechanism of protection conferred by early full-term pregnancy. In order to elucidate the molecular pathways through which pregnancy exerts a protective effect, we have analyzed the genomic profile of Lob 1 present in reduction mammoplasty specimens obtained from parous and nulliparous postmenopausal women. The genes differentially expressed are related to immune-surveillance, DNA repair, programmed cell death, transcription, and chromatin structure/activators/co-activator. In the present study we performed real-time RT-PCR using a low-density array or a microfluid card for genes related to the immune system and programmed cell death, using 18S as an internal control [TaqMan(R) Low Density Array Human Immune Panel (Applied Biosystems)]. Breast epithelial cells from parous women significantly overexpressed 17 out of 20 genes (p<0.001) with respect to the nulliparous breast. BCL2-associated X protein, Complement component 3, CD45 antigen, glyceraldehyde-3-phosphate dehydrogenase, granulysin, and chemokine (C-C motif) ligand 19 were expressed more than 30-fold with respect to nulliparous breast cells. Only three out of 20 genes [selectin P (granule membrane protein 140 kDa, antigen CD62), Fas (TNF receptor superfamily, member 6) and chemokine (C-X-C motif) ligand 11], were downregulated in parous breast with respect to nulliparous breast cells. The data lead us to conclude that an early pregnancy, by shifting the Stem cell 1 to Stem cell 2, makes the latter more easily recognized by the immune-surveillance system, which initiates the programmed cell death pathway if exposure to toxic or carcinogenic agents occurs.

Detection of chromosomal aberrations by comparative genomic hybridization during transformation of human breast epithelial cells in vitro.

Breast cancer is the most frequent malignancy in women. It is well recognized that tumorigenesis is a multistep process resulting from the accumulation of sequential genetic alterations. In breast cancers LOH has been described on one or both arms of multiple chromosomes. Comparative genomic hybridization (CGH) analysis was performed to identify chromosomal imbalances in the breast epithelial cells (HBEC). We have used a human in vitro-in vivo system in which the environmental carcinogen benz(a)pyrene (BP) and the c-Ha-ras oncogene were utilized for inducing in vitro transformation of HBEC. Immortal MCF-10F cells were treated with BP which resulted in the transformed cell line BP-1 that was further enhanced by transfection with the c-Ha-ras to generate the cell line BP-1-Tras. This cell line is tumorigenic when injected in severe combined immunodeficient (SCID) mice, generating the tumor cell line BP-1-Tras T J#4. Our comparative genomic hybridization analysis indicates that the most overrepresented segment after cell transformation and in the BP-1, BP-1-Tras and in the tumor cell line were 1p (80%), 5q21-ter (80%), 8q24.1 (90%) and Xq27-28 (60%). DNA sequence amplification at 10p14-15 was observed in BP-1-Tras T J#4 cells. Allelic losses of chromosome 4, 8p11-21 and 15q11-12, occur after cell transformation and are maintained consistently during tumorigenesis.

Carcinogenicity of estrogens in human breast epithelial cells.

Epidemiological and clinical evidences indicate that breast cancer risk is associated with prolonged ovarian function that results in elevated circulating levels of steroid hormones. Principal among these is estrogen, which is associated with two important risk factors, early onset of menarche and late menopause. However, up to now there is no direct experimental evidence that estrogens are responsible of the initiation of human breast cancer. We postulate that if estrogens are causative agents of this disease, they should elicit in human breast epithelial cells (HBEC) genomic alterations similar to those exhibited by human breast cancers, such as DNA amplification and loss of genetic material representing tumor suppressor genes. These effects could result from binding of the hormone to its nuclear receptors (ER) or from its metabolic activation to reactive metabolites. This hypothesis was tested by treating with the natural estrogen 17beta-estradiol (E2) and the synthetic steroid diethylstilbestrol (DES) MCF-10F cells, a HBEC line that is negative for ER. Cells treated with the chemical carcinogen benzo (a) pyrene (BP) served as a positive control of cell transformation. BP-, E2-, and DES-treated MCF-10F cells showed increases in survival efficiency and colony efficiency in agar methocel, and loss of ductulogenic capacity in collagen gel. The largest colonies were formed by BP-treated cells, becoming progressively smaller in DES- and E2-treated cells. The loss of ductulogenic capacity was maximal in BP-, and less prominent in E2- and DES-treated cells. Genomic analysis revealed that E2- and DES-treated cells exhibited loss of heterozygosity in chromosomes 3 and 11, at 3p21, 3p21-21.2, 3p21.1-14.2, and 3p14.2 14.1, and at 11q23.3 and 11q23.1-25 regions, respectively. It is noteworthy that these loci are also affected in breast lesions, such as ductal hyperplasia, carcinoma in situ, and invasive carcinoma. Our data are the first ones to demonstrate that estrogens induce in HBEC phenotypic changes indicative of cell transformation and that those changes are associated with significant genomic alterations that might unravel new pathways in the initiation of breast cancer.

Cancer risk related to mammary gland structure and development.

The breast undergoes dramatic changes in size, shape, and function in association with growth, reproduction, and post-menopausal regression. Those changes impact women's lifetime breast cancer risk. An early first full-term pregnancy exerts a protective effect, emphasizing the need for understanding the role of reproductive influences on breast development and on cancer initiation and progression, and providing a paradigm for developing preventive strategies based on physiological principles. Even though the cause of breast cancer and the ultimate mechanisms through which an early pregnancy protects from cancer development remain largely unknown, a likely explanation for this protection has been provided by experimental in vivo and in vitro models. These studies have led to the conclusions that cancer initiation requires the interaction of a carcinogen with an undifferentiated and highly proliferating mammary epithelium, whereas differentiation of the mammary gland inhibits carcinogenic initiation. The process of mammary gland differentiation is the result of complex interactions of ovarian, pituitary, and placental hormones, which in turn induce inhibition of cell proliferation, downregulation of estrogen and progesterone receptors, activation of specific genes, such as inhibin, mammary derived growth factor inhibitor and a serpin-like gene, and expression of extracellular matrix proteins in the normal breast. Cell immortalization and transformation are associated with the expression of ferritin H and S100P protein, which serve as markers of cancer initiation. Comparative studies of normal and neoplastic breast development have unraveled similarities with experimental models that validate the extrapolation of findings for testing hypotheses on the initiation and progression of breast cancer.

The pathway of neoplastic transformation of human breast epithelial cells.

The morphological analysis of breast cancer development indicates this to be a multistep process that progressively evolves from ductal hyperplasia and atypical ductal hyperplasia, which represent the initial stages of neoplastic growth, to carcinoma in situ, invasive carcinoma, and ultimately metastasis, as has been documented for a number of other malignancies. The understanding of the cellular and molecular processes that lead a normal cell to malignancy requires the analysis of pure populations of human breast epithelial cells (HBEC) representing specific stages of neoplastic progression. The neoplastic transformation of HBEC in vitro represents a successful model for obtaining knowledge about the molecular and biological alterations that may contribute to the tumorigenic mechanisms. We present here a current understanding of chemically transformed HBEC in the following aspects: (1) factors affecting the transformation of HBEC such as immortalization; (2) new targets for studying the mechanism of cell immortalization such as alterations in telomerase activity, differential expression of cell cycle-dependent genes, and others recently isolated through differential cloning, such as H-ferritin, and a calcium binding protein; (3) genetic mechanisms underlying cell transformation; and (4) application of the microcell-mediated chromosome transfer technique as an approach to testing the functional role of specific genes whose dysregulation or loss of function may contribute to the ultimate cell transformation. Further efforts in this cell system will be directed to determining the roles of identified molecular changes as well as the mapping/cloning of tumor suppressor or senescence genes.

Mammary gland architecture as a determining factor in the susceptibility of the human breast to cancer.

The developmental pattern of the breast can be assessed by determining the composition of the breast in specific lobular structures, which are designated as lobules type 1 (Lob 1), lobules type 2 (Lob 2), and lobules type 3 (Lob 3), with Lob 1 being the less developed and Lob 3 being the most differentiated or with the highest number of ductules per lobular unit. In the present work, the patient population consisted of three groups of women who underwent surgical procedures: The first group included women who underwent reduction mammoplasty (RM) for cosmetic reasons. The second group included women who underwent prophylactic subcutaneous mastectomy after genetic counseling for either carrying the BRCA-1 gene or belonging to a pedigree with familial breast cancer (FAM), and the third group included women who underwent modified radical mastectomy (MRM) for the diagnosis of invasive carcinoma. The RM group consisted of 33 women, of whom 9 were nulliparous and 24 were parous. The FAM group consisted of 17 women, of whom 8 were nulliparous and 9 were parous. The MRM group consisted of 43 women, of whom 7 were nulliparous and 36 were parous. The analysis of the lobular composition of all of the samples from the RM group, which is considered the control group, revealed that Lob 1 represented 22%, Lob 2 represented 37%, and Lob 3 represented 38%, whereas the tissue examined from the FAM and MRM groups contained a preponderance of Lob 1 at 48% and 74%, respectively, over Lob 3, which was 10% and 3%, respectively. When the results of the analysis of breast tissue were separated according to the pregnancy history of the donor, it was found that in the control group or RM, there was a significant difference in lobular composition. Nulliparous women of the RM group showed a preponderance of Lob 1 (46%) over parous women, which contained only 17%, whereas the percentage of Lob 3 in the nulliparous group was significantly lower (7%) than the parous group (48%). In the breast tissues obtained from FAM and MRM, no significant differences in lobular composition were observed, as all of the samples contained a higher concentration of Lob 1, independent of the pregnancy history. The breast tissue of FAM and MRM of parous women had a developmental pattern that was similar to that of nulliparous women of the same group and that was less developed than the breast of parous women of the control group. An important difference between the Lob 1 of the FAM group versus the control (RM) and the MRM group was that most of these lobules had thin ductules with an increase in hyalinization of the intralobular stroma manifested in the whole-mount preparation as an alteration in the branching pattern. The data suggest that the breast tissue of women with invasive cancer, as well as those from a background of familial breast cancer, have an architectural pattern different from the control or normal tissues and that the BRCA-1 or related genes may have a functional role in the branching pattern of the breast during lobular development, mainly in the epithelial stroma interaction.

Developmental, cellular, and molecular basis of human breast cancer.

Breast cancer, which is the most common neoplastic disease in females and accounts for up to one third of all new cases of women's cancer in North America, continues to rise in incidence. In addition, the mortality caused by this disease has remained almost unchanged for the past 5 decades, becoming only second to lung cancer as a cause of cancer-related death. The failure in eradicating this disease is largely due to the lack of identification of a specific etiologic agent, the precise time of initiation, and the molecular mechanisms responsible for cancer initiation and progression. Despite the numerous uncertainties surrounding the origin of cancer, there is substantial evidence that breast cancer risk relates to endocrinologic and reproductive factors. The development of breast cancer strongly depends on the ovary and on endocrine conditions modulated by ovarian function, such as early menarche, late menopause, and parity. However, the specific hormone or hormone combinations responsible for cancer initiation have not been identified, and their role as protective or risk factors is still incompletely understood. A highly significant female hormone is estrogen, which is involved in the development of a variety of cancers, but it is still unclear whether estrogens are carcinogenic to the human breast. An understanding of whether estrogens cause mutations, and, if so, whether they act through hormonal effects activated by receptor binding, cytochrome P450-mediated metabolic activation, or compromise the DNA repair system, is essential for determining whether this steroid hormone is involved in the initiation or progression of breast cancer. This knowledge has to be based on a multidisciplinary approach encompassing studies of the development of the breast, influence of hormones on the differentiation of individual structures, and their interrelations in the pathogenesis of breast cancer. The analysis of the mechanisms involved would require confirmation in the adequate in vitro models and determination of the role played by genomic alterations in both cancer initiation and progression.

Hormonal approach to breast cancer prevention.

Breast cancer is more frequent in nulliparous women, while its incidence is significantly reduced by full-term pregnancy. The fact that the protection conferred by pregnancy is observed in women from different countries and ethnic groups, regardless of the endogenous incidence of this malignancy, indicates that this protection does not result from extrinsic factors specific to a particular environmental, genetic, or socioeconomic setting, but rather from an intrinsic effect of parity on the biology of the breast. Using an experimental system we have shown that treatment of young virgin rats with human chorionic gonadotropin (hCG), like full-term pregnancy, efficiently inhibits the initiation and progression of chemically induced mammary carcinomas. Treatment of young virgin rats with hCG induced a profuse lobular development of the mammary gland, reduced the proliferative activity of the mammary epithelium, and induced the synthesis of inhibin, a secreted protein with tumor-suppressor activity. HCG treatment also increased the expression of the programmed cell death (PCD) genes testosterone repressed prostate message 2 (TRPM2), interleukin 1-beta-converting enzyme (ICE), p53, c-myc, and bcl-XS, induced apoptosis, and downregulated cyclins. PCD genes were activated through a p53-dependent process, modulated by c-myc, and with partial dependence on the bcl-2 family-related genes. The possibility that this hormonal treatment activates known or new genes was tested by differential display technique. We have identified a series of new genes, hormone-induced-1 (HI-1) among them. The characterization of their functional role will contribute to clarify the mechanisms through which hCG inhibits the initiation and progression of mammary cancer. Of great significance was the observation that PCD genes remained activated even after lobular formations had regressed due to the cessation of hormone administration. We postulate that this mechanism plays a major role in the long-lasting protection exerted by hCG from chemically induced carcinogenesis, and might be also involved in the lifetime reduction in breast cancer risk induced in women by full-term pregnancy. The implications of these observations are two-fold: on one hand, they indicate that hCG, as pregnancy, may induce early genomic changes that control the progression of the differentiation pathway, and on the other, that these changes are permanently imprinted in the genome, regulating the long-lasting refractoriness to carcinogenesis. The permanence of these changes, in turn, makes them ideal surrogate markers of hCG effect in the evaluation of this hormone as a breast cancer preventive agent.

S100P calcium-binding protein overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo.

The mechanism of cell immortalization of human breast epithelial cells leading to neoplastic transformation is not clear. The isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10F, have provided a valuable tool to identify genes involved in this process. Using the technique of differential display, we have identified seven cDNA bands differentially displayed in the MCF-10F cells when compared with the mortal S130 cells from which MCF-10F was originated. One of these bands was isolated and cloned. Sequence analysis revealed 99% homology to the EF-hand calcium-binding protein S100P (Placental). The clone was overexpressed in the immortal cell line MCF-10F when compared to the mortal counterpart S130 or other primary cultures of human breast epithelial cells. In addition, it was highly expressed in chemically transformed breast epithelial cell lines (BP1E and D3. 1), breast cancer cell line T47D, as well as in three invasive ductal carcinomas when compared to their normal adjacent tissue. The S100P protein was localized by immunohistochemistry, using a monoclonal antibody against the same amino acid sequence of the gene cloned, in ductal hyperplasias, in situ and invasive ductal carcinoma, but not in the normal tissues. We concluded that S100P overexpression is an early event that might play an important role in the immortalization of human breast epithelial cells in vitro and tumor progression in vivo.

Atlas and histologic classification of tumors of the rat mammary gland.

Studies performed in experimental animal models have demonstrated that mammary cancer is a complex multistep process that can be induced either by chemicals, radiation, viruses, or genetic factors. Rodent models have been useful for dissecting the initiation, promotion, and progression steps of mammary carcinogenesis. Chemically induced mammary tumors, such as those induced by 7,12-dimethylbenz(a)anthracene and N-methyl-N-nitrosourea, are, in general, hormone-dependent adenocarcinomas whose incidence, number of tumors per animal, tumor latency, and tumor type are influenced by the age, reproductive history, and endocrinologic milieu of the host at the time of carcinogen exposure as well as by diet and the dose of carcinogen administered. There is a need to classify tumors according to their histopathological type because those characteristics have implications in the interpretation of experimental data. In the classification presented here we attempt to provide a working framework for diagnosis of the type of lesions found in the mammary glands of rats treated with chemical carcinogens or radiation and to clarify criteria for establishing the basic biological characteristics of tumors.

Cellular basis of breast cancer susceptibility.

Breast cancer originates in undifferentiated terminal structures of the mammary gland. The terminal duct of the Lob 1 of the human female breast is the site of origin of ductal carcinomas. Cell replication and the concentration of estrogen receptors type at in Lob 1 are at their peak during early adulthood, at a time during which the breast is more susceptible to carcinogenesis, decreasing considerably with aging. More importantly, when treated with carcinogens in vitro they express phenotypes indicative of cell transformation. These studies indicate that in humans there is a target cell of carcinogenesis, which is found in a specific compartment whose characteristics are a determinant factor in the initiation event. These target cells will become the stem cells of the neoplastic event, depending upon: a) topographic location within the mammary gland tree, b) age at exposure to a known or putative genotoxic agent, and c) reproductive history of the host. Epidemiological findings such as the higher incidence of breast cancer in nulliparous women and in women having early menarche support this concept, because it parallels the higher cancer incidence elicited by carcinogens when exposure occurs at a young age. In addition, it has been shown that increase in parity is associated with a pronounced decrease in the risk of breast cancer, each additional live birth conferring a 10% risk reduction. Thus, the protection afforded by early full-term pregnancy in women could be explained by the higher degree of differentiation of the mammary gland at the time in which an etiologic agent or agents act. The relevance of our work lies in the side by side comparison of in vivo and in vitro studies in the human breast that validates experimental data for extrapolation to the human situation. The finding that cell proliferation is of importance for cancer initiation, whereas differentiation is a powerful inhibitor, provides novel tools for developing rational strategies for breast cancer prevention and control.

Functional roles of chromosomes 11 and 17 in the transformation of human breast epithelial cells in vitro.

Genomic alterations in primary breast cancer play a role in the initiation and progression of the disease. We have analyzed the molecular events involved in the initiation and progression of the neoplastic process in an in vitro experimental system. Immortalization of human breast epithelial cells (HBEC) is associated with 3:9 translocation, p53 mutation and microsatellite instability (MSI) of chromosomes 11p13, and 17p. BP1-E cells, derived from the immortalized MCF-10F cells transformed by the carcinogen benzo(a)pyrene (BP), express in vitro growth advantage, anchorage independence, enhanced chemoinvasiveness, loss of ductulogenic capabilities and tumorigenesis in a heterologous host. This neoplastic progression is also associated with mutations and/or amplification of c-H-ras, int-2, c-neu, c-myc and MDM2, MSI at 11q25 and 13q12-q13 and loss of heterozygosity at 17p. In order to test whether chromosomes 11 or 17 play a functional role in the phenotypic expression of transformation of BP1E cells, we utilized microcell-mediated chromosome transfer (MMCT) technique for inserting the corresponding normal chromosomes to these transformed cells. BP1E cells were transfected with PsV2neo plasmid and fused with microcells obtained from the mouse cell line A9, containing a normal chromosome 11 or 17 (A9-11neo and A9-17neo cells, selected in G418 and cloned. Sixteen primary microcell hybrids from each chromosome transfer, designated BP1E-11neo and BP1E-17neo survived selection in G-418 containing medium. A single clone from each group, BP1E-11neo #145 and BP1E-17neo D100, survived subcloning and were utilized for a detailed panel of analyses. The presence of a donor chromosome was confirmed by dual color fluorescence in situ hybridization (FISH), southern blot analysis of the marker vector pSV2neo, and microsatellite polymorphism analysis. The transfer of the normal chromosomes 11 and 17 resulted in a 50% and 90% inhibition of cell growth respectively, and reduced both colony efficiency and colony size. Telomerase activity was significantly reduced only by chromosome 17 insertion, providing a possible explanation for the more significant senescence observed in BP1E-17neo D100 cells. Microsatellite polymorphism analysis revealed that three loci, 11q13-23, 11q23.1, and 11q23.3 (markers D11S911, DRD2, and D11S29) were retained in BP1E-11neo #145 cells, and two, 17q24.2-25.2, 17q25.2 (markers D17S515 and D17S785 were retained in BP1E-17neo D100 cells. We conclude that the specific regions of normal chromosomes 11 and 17 transferred play a functional role in the expression of immortal and transformed phenotypes of HBEC in vitro.

Allelic instability as a predictor of survival in Egyptian breast cancer patients.

This work was designed with the purpose of determining whether the presence of allelic imbalances (AI) such as microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 2, 11, 13, and 17 in primary breast cancer could be used as prognostic indicators of patient survival. The DNA from breast cancers removed from 29 patients who were followed-up for up to five years was analyzed for MSI and LOH using a panel of 24 markers located at chromosome 2 (TPO, D2S131, D2S144, D2S171, D2S177, D2S119, D2S123, D2S147 and D2S136), chromosome 11 (C-RAS, Int-2, D11S940, D11S912), chromosome 13 (D13S289, D13S260, D13S267, D13S218, D13S263, D13S155, and D13S162), and chromosome 17 (D17S513, TP53, D17S855, and D17S785). The frequency of AI in the markers studied ranged from 30-55%, being highest for D11S912, D2S171, TP53 and D17S855. Univariate analysis showed association between overall survival rate and AI in 9 out of the 24 markers tested. Five of them were located at the area of the mismatch repair gene (MMR)-2 gene, two at 11p, one at 13q and one at 17p. Using multivariate analysis, it was observed that only pathological and clinical stage (defined as stage II or not) and AI at D2S171, D11S912, or D17STP53 generated significant predictive models for survival.

Inhibition of rat mammary tumorigenesis by human chorionic gonadotropin associated with increased expression of inhibin.

This work was designed to test whether the suppression of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinomas by human chorionic gonadotropin (hCG) is associated with the synthesis of inhibin. For this purpose, virgin rats received 8 mg DMBA/100 g body weight; 20 d later they were injected daily with 100 IU of hCG for 40 d (DMBA+hCG group). Age-matched untreated (control), hCG-, and DMBA+saline-treated rats were used for comparisons. Mammary tissues were collected for histopathological and mRNA analyses after 5, 10, 20, and 40 d of hCG injection and 20 d after treatment. None of the animals in the control and hCG-treated groups developed mammary tumors. DMBA-treated rats developed a high incidence of both microscopic lesions, i.e., intraductal proliferations and ductal carcinomas in situ, and palpable tumors. In DMBA+hCG-treated rats, the incidence of microscopic and palpable tumors was markedly reduced. In these animals, alpha- and beta-inhibin immunoreactivity was elevated in the non-tumoral mammary glands in association with lobule formation and in the tumors. Inhibin A and B mRNAs were also elevated in the mammary tissue, and c-myc and c-jun were induced by the hormonal treatment. DMBA alone did not modify the expression of these genes. Our findings indicate that inhibin production and gene activation are associated with both mammary gland differentiation and tumor regression.

Pattern of distribution of cells positive for estrogen receptor alpha and progesterone receptor in relation to proliferating cells in the mammary gland.

Since cell proliferation is indispensable for the growth and development of the breast, and estrogens are considered to play a major role in promoting cell proliferation, while progesterone influences its differentiation, the present work was designed with the purpose of verifying the relationship between cells containing steroid hormone receptors and proliferating cells in the normal human breast. Twelve breast samples were analyzed for their content of lobules type 1 (Lob1), Lob2, Lob3, and Lob4, and the number of cells containing estrogen receptor alpha (ER-alpha), progesterone receptor (PgR), or expressing Ki67 antibody was determined by double immunocytochemical technique with specific antibodies. The highest percentage of ER-alpha, PgR, and Ki67 positive cells was found in Lob1, with a progressive reduction in the more differentiated Lob2 and Lob3. ER-alpha and PgR positive cells were found exclusively in the breast epithelium and were negative for Ki67, while cells positive for Ki67 did not express receptors. These findings were compared with the distribution of ER-alpha and PgR in the autoradiographs of mammary gland of young virgin rats inoculated with 3H-thymidine for determination of the DNA labeling index (DNA-LI). Both the DNA-LI and the percentage of ER-alpha and PgR positive cells were maximal in the epithelium of terminal end buds, and these values were reduced in alveolar buds and lobules. ER-alpha and PgR positive cells did not proliferate, and those cells that had incorporated 3H-thymidine were negative for both receptors. Our results led us to conclude that the content of ER-alpha and PgR in the normal mammary tissue varies with the degree of lobular development, in parallel with cell proliferation. However, the expression of receptors occurs in cells other than the proliferating cells, indicating that they represent at least two separate cell populations. These findings open new avenues towards the understanding of the mechanisms through which estrogens and progesterone affect the proliferative activity of breast epithelial cells, and their role in the initiation of the cascade of events that leads a normal cell to cancer.

Roscovitine induces cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells.

We have previously shown (Mgbonyebi et al., Anticancer Res., 18: 751-756, 1998) that roscovitine, an olomoucine-related purine analogue and a selective inhibitor of cyclin-dependent kinases, inhibited the proliferative activity of human breast epithelial cells in vitro. The purpose of the present study was to identify the cellular processes and targets affected by roscovitine treatment in the estrogen receptor-negative MDA-MB-231 human breast carcinoma cells. Treatment of the cells with 10 microg/ml roscovitine daily for a length of time ranging from 24 to 240 h revealed that the compound inhibited DNA synthesis, induced cell death, and irreversibly inhibited the proliferative activity of the cells. Morphological analysis of roscovitine-treated cells by light and fluorescence microscopy demonstrated that this cyclin-dependent kinase inhibitor induced cell shrinkage, chromatin condensation, reorganization of actin microfilament architecture, and extensive detachment of cells from the cell culture substratum. These cellular events are all known to be associated with apoptosis. Collectively, the data generated from this study suggest that roscovitine induced apoptosis in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Because the efficacy of many anticancer drugs depends on their ability to induce apoptotic cell death, modulation of this parameter by roscovitine may provide a new chemopreventive and chemotherapeutic strategy for the clinical management of hormone-resistant breast cancers.

Microsatellite instability during the immortalization and transformation of human breast epithelial cells in vitro.

The objective of this study was to determine whether microsatellite instability (MSI) and loss of heterozygosity (LOH) are involved in the immortalization of human breast epithelial cells (HBECs) in vitro and in the early stages of their transformation by benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA). We performed a genome-wide analysis of a total of 466 microsatellite DNA polymorphism loci along the X chromosome and the 22 pairs of human autosomes. MSI was found in the immortalized MCF-10F cells at the following loci: D11S1392 (on chromosome 11p13) and D17S849 (at 17p13.3), D17S796 (at 17p13.1), D17S513 (at 17p13.1), TP53 (at 17p13.1), D17S786 (at 17p13.1), and D17S520 (at 17p12) on chromosome 17. The BP-transformed cells exhibited MSI in the same loci and also in locus D11S912 (at 11q25). The more transformed BP1E cells also exhibited MSI on chromosome 13q12-13 at D13S260 and D13S289, markers known to flank the breast cancer susceptibility gene BRCA2. In the DMBA-transformed D3 and D3-1 cells, MSI was observed at the locus D13S260 in addition to the previously reported locus D16S285 (at 16q12.1). No LOH was observed on any of the chromosomes tested in these cells. These observations led us to conclude that the immortalization and transformation of HBECs may involve defects in mechanisms responsible for the cell's genomic stability, such as DNA replication and DNA mismatch repair.