In vitro transformation of human congenital naevus to malignant melanoma.

The incidence of melanoma is estimated to be growing at the second fastest rate among all cancers in the United States. The progression of the melanocyte to a malignant melanoma involves various sequential steps: development of benign naevocellular naevus, preneoplastic dysplastic naevus, primary melanoma, and metastatic melanoma. Despite these clearly defined stages, very little is known about the molecular events leading to melanoma progression. We established a human congenital naevus cell line (UISO-CMN-1). UISO-CMN-1 cells were confirmed to have melanocytic origin by S100 immunoreactivity and the presence of melanin granules and melanosomes. UISO-CMN-1 cells, even though they showed structural and numerical abnormalities in karyotype, were non-tumorigenic when transplanted into athymic mice. However, following frequent exposure to ultraviolet C radiation, UISO-CMN-1 cells acquired tumorigenic potential. Transformation of UISO-CMN-1 cells into tumorigenic cells was accompanied by induction of ganglioside-2 expression without any significant changes in cellular ganglioside-3. These transformed and non-transformed UISO-CMN-1 cell lines can serve as excellent research tools for studying the molecular changes associated with melanoma development and progression, and for identifying agents that might prevent development of malignant melanoma.

Bcl-2 and Bax are differentially expressed in hyperplastic, premalignant, and malignant lesions of mammary carcinogenesis.

Previously, we found that vorozole (Vz), a nonsteroidal aromatase inhibitor, suppresses the development and progression of mammary tumors in rats. Here we evaluated for the first time the expression of cell death-related proteins Bcl-2 and Bax in hyperplastic, premalignant (carcinoma in situ), or malignant (carcinoma) lesions of mammary carcinogenesis; we also assessed whether these proteins are involved in mediating Vz-induced cell death in tumors. We found that Bcl-2 and Bax were equally expressed in epithelial cells of terminal end buds, ducts, and alveoli. However, in myoepithelial cells, the level of Bax expression was much higher than the level of Bcl-2 expression. Bcl-2 and Bax levels in hyperplastic lesions were similar to those of normal mammary epithelial cells but lower in most carcinomas in situ and carcinomas. In animals with established mammary tumors, Vz induced apoptotic cell death, which was primarily associated with a decrease in Bcl-2 and, to a lesser extent, with a decrease in Bax. These data support the hypothesis that Bcl-2 loss is more potent than Bax gain in regulating apoptotic cell death in mammary tumors.

Induction of differentiation by 1alpha-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors.

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).

Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells.

The present study was undertaken to determine if (a) genistein induces topo II-mediated DNA damage in HT-29 colon cancer cells; and (b) if this damage is required to induce apoptosis. DNA damage was evaluated using the comet assay. Apoptosis was determined by the ethidium bromide/acridine orange staining technique. DNA breakage was noted within 1 h of treatment. Apoptosis was only induced with high concentrations (>/=60 microM) of genistein. Marked inhibition of HT-29 cell growth was evident at concentrations ranging from 60 to 150 microM. This was associated with a cell cycle arrest at G(2)/M. Similar findings were obtained in SW-620 and SW-1116 colon cancer cell lines. Aclarubicin, a topo II antagonist, reduced genistein-induced DNA breaks but did not reduce apoptosis. These data suggest that, in colon cancer cells, topo II serves as the enzymatic target of genistein. Furthermore, topo II-mediated DNA cleavage is not required for the induction of apoptosis.

Differentiation of human breast carcinoma cells by a novel vitamin D analog: 1alpha-hydroxyvitamin D5.

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3, can induce differentiation in breast cancer cells; however, it is hypercalcemic in vivo. Therefore, development of non-calcemic analogs of vitamin D has received considerable attention. Recently, we synthesized an analog of vitamin D [1alpha(OH)D5] that exhibits much less calcemic activity than 1alpha,25-dihydroxyvitamin D3. In this study, we evaluated the cell-differentiating action of 1alpha(OH)D5 in breast cancer cells. Following 10 days treatment with 1alpha(OH)D5 [(10-7 M) in UISO-BCA-4], we observed induction of intracytoplasmic casein, intracytoplasmic lipid droplets, ICAM-1, nm23, and specific biomarkers associated with breast cell differentiation. 1alpha(OH)D5 treatment also showed induction of vitamin D receptor and TGFbeta1 proteins. UISO-BCA-4 cells pretreated for 10 days in vitro with 1 microM 1alpha(OH)D5 failed to form tumors when transplanted into athymic mice. Similarly, 4 and 8 ng 1alpha(OH)D5 treatment three times weekly inhibited the growth of UISO-BCA-4 cells injected into athymic mice. These results suggest that this new vitamin D analog may be of significant therapeutic value for breast cancer.

Cytostatic mechanism and antitumor potential of novel 1H-cyclopenta[b]benzofuran lignans isolated from Aglaia elliptica.

A total of five 1H-cyclopenta[b]benzofuran lignans (1-5) isolated from the stems of Aglaia elliptica B1. (Meliaceae) inhibited the growth of human cancer cells in culture. Of particular note, the IC50 values observed with 1 (methyl rocaglate), 2 (4'-demethoxy-3',4'-methylenedioxy-methyl rocaglate) and 5 (1-O-formyl-4'-demethoxy-3',4'-methylenedioxy-methyl rocaglate) were in the 1-30 ng/ml range. Prompted by the high potency of these responses, additional studies were performed with 2, a structurally representative isolate that was available in sufficient quantity as a result of the isolation process. Utilizing cultured Lu1 (human lung carcinoma) cells as a model, compound 2 induced accumulation in the G1/G0 phase of the cell cycle after 24 or 32 h of incubation; normal cell-cycle dynamics were observed at subsequent time periods. Cell proliferation was inhibited in a dose-dependent manner, but during the course of wash-out experiments, colony formation was not reduced. In addition, as judged by [3H]leucine incorporation, the test compound strongly inhibited protein biosynthesis (IC50 = 25 ng/ml). In analogous studies, nucleic acid biosynthesis was not reduced, even when cells were treated with concentrations as high as 1 microg/ml. These data suggest inhibition of protein synthesis is a key mode of action, and the compound functions by a cytostatic mechanism. Utilizing a human breast cancer cell line (BC1) sensitive to compound 2 in culture (IC50 = 0.9 ng/ml), an initial assessment of antitumor potential was performed. In accord with the in vitro results, the growth of BC1 in athymic mice was delayed by treatment with compound 2 (10 mg/kg body weight, three times per week, i.p.). Body weight was unaffected and no signs of overt toxicity were observed. However, growth paralleled that of the control group at later time points. Thus, novel 1H-cyclopenta[b]benzofuran lignans are potent cytostatic inhibitors of protein biosynthesis and are capable of delaying tumor growth in an in vivo model. Their full clinical or basic utility requires further investigation.

Genistein induces maturation of cultured human breast cancer cells and prevents tumor growth in nude mice.

Results of recent studies in animal models of mammary carcinogenesis showed that the soybean isoflavone genistein is a chemopreventive agent. The objective of the present study was to determine whether soybean isoflavones can be used for the prevention of human breast carcinogenesis. Human adenocarcinoma cells that are either estrogen-receptor positive (such as MCF-7) or estrogen-receptor negative (such as MDA-MB-468) were used as our model system. Treatment of these cells with genistein concentrations of 15, 30, and 45 micromol/L resulted in cell growth inhibition, which was accompanied by the expression of maturation markers. Maturation was monitored by the induction of intracytoplasmic casein and lipids and the membrane protein intercellular adhesion molecule-1. These maturation markers were optimally expressed after 9 d of treatment with 30 mmol genistein/L. Both estrogen receptor-positive and -negative cells became differentiated in response to genistein treatments, suggesting that the antiestrogenic function of genistein is unrelated to the mechanism of cell differentiation. Daidzein, the other major isoflavone component of soybeans, did not induce differentiation in either MCF-7 or MDA-MB-468 cells. To explore the potential applications of this result, we used the nude mouse xenograft model of carcinogenesis. Treatment of either cell line with genistein before implantation into nude mice diminished the cells' tumorigenic potential. These data suggest that initiation of the differentiation program provides a protective effect against tumor growth in mouse xenografts.

Metabolism of N-[4-hydroxyphenyl]retinamide (4-HPR) to N-[4-methoxyphenyl]retinamide (4-MPR) may serve as a biomarker for its efficacy against human breast cancer and melanoma cells.

A clinical trial of N-[4-hydroxyphenyl]retinamide (4-HPR) has been in progress for the past 4 years to evaluate its role in chemoprevention of breast cancer. However, it is currently not known whether the effect of 4-HPR in breast cells is mediated by 4-HPR directly or through one of its metabolites. In this report, we investigated in vivo and in vitro effects of 4-HPR on three different breast carcinoma cells and two different melanoma cell lines. In vitro, the growth of all three breast carcinoma cell lines was inhibited by 4-HPR. Only one of two melanoma cell lines (UISO-Mel-1) showed growth inhibition to 4-HPR. The cell lines sensitive to 4-HPR in vitro also showed inhibition to 4-HPR in a xenograft model. Dietary 4-HPR (0.5 mmol/kg diet) reduced the growth of UISO-BCA-1 xenografts in female athymic mice, but had no effect on UISO-Mel-6 xenografts. Metabolism investigations of the 4-HPR-sensitive and insensitive cell lines indicated that N-[4-methoxyphenyl]retinamide (4-MPR), the major metabolite of 4-HPR, was detected only in cells sensitive to 4-HPR. Further in vitro studies with 4-MPR suggested that it is not an active metabolite of 4-HPR as it failed to inhibit growth of 4-HPR-resistant UISO-Mel-6 cells, and showed no dose-dependent inhibition of 4-HPR-sensitive breast carcinoma and melanoma cell lines. Our results in the present study indicate that, although 4-MPR is not an active metabolite of 4-HPR, detection of this metabolite in the malignant cells may serve as an indirect biomarker to predict response of cells to 4-HPR.

Plasma c-erbB-2 levels in breast cancer patients: prognostic significance in predicting response to chemotherapy.

PURPOSE: To determine the significance of plasma c-erbB-2 levels to assess the extent of disease spread and to predict the response to chemotherapy in node-positive breast cancer patients. METHODS: We determined plasma levels of c-erbB-2 in 79 stages II and III breast cancer patients who received cyclophosphamide, methotrexate, and flourouracil (CMF)/cyclophosphamide, methotrexate, fluorouracil, vincristine, and prednisone (CMFVP) chemotherapy. All patients had a minimum follow-up of greater than 60 months or until disease recurrence. Plasma samples were obtained before and after chemotherapy. Plasma c-erbB-2 levels were quantified by enzyme-linked immunoassay. c-erbB-2 levels were analyzed in relation to the patients' axillary lymph node status, menopausal status, disease status, disease-free survival (DFS), and steroid receptor status of tumor. RESULTS: Plasma c-erbB-2 levels varied widely in breast cancer patients. In general, when all patients were included in the analyses, plasma c-erbB-2 levels before chemotherapy correlated significantly with the number of positive axillary lymph nodes and with postchemotherapy c-erbB-2 levels. No association was observed between pre- or postchemotherapy c-erbB-2 levels and other variables (patients' age at diagnosis, receptor status of the tumor, or disease status). The prognostic significance of different factors (ie, nodal status [one to three v > three positive nodes], menopausal status [pre- v postmenopausal women], estrogen receptor [ER] status [ER+ v ER-], and pre- and postchemotherapy c-erbB-2 levels) in predicting DFS was determined in all study patients. Among the variables examined, nodal status was the strongest predictor of DFS in these patients. The second most significant prognostic marker was postchemotherapy c-erbB-2 level. Prechemotherapy c-erbB-2 levels showed prognostic significance for DFS in a subset of breast cancer patients (ie, patients with > three positive nodes). Patients with greater than three positive lymph nodes and those with greater than 100 fmol/mL of plasma c-erbB-2 levels before therapy had significantly shorter DFS than did those patients with 100 fmol/mL or less c-erbB-2 levels. CONCLUSION: In breast cancer patients, determination of c-erbB-2 levels before therapy is an important biomarker to assess the extent of disease spread in the lymph nodes. Postchemotherapy c-erbB-2 levels are also a prognostic indicator for DFS in patients who receive chemotherapy. Finally, in a subgroup of patients with greater than three positive nodes, prechemotherapy c-erbB-2 levels are a prognostic marker for response of patients to standard chemotherapy.

Development of a new metastatic human breast carcinoma xenograft line.

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.

nm23--relationship to the metastatic potential of breast carcinoma cell lines, primary human xenografts, and lymph node negative breast carcinoma patients.

BACKGROUND: Since the discovery of nm23 (nonmetastatic) by Steeg et al. in 1988, a number of tumor cohort studies have shown an inverse relationship between the levels of expression of the nm23-H1 protein and disease aggressiveness and tumor metastatic potential. METHODS: The relationship between the expression of nm23 protein and the metastatic potential of human breast carcinoma was analyzed in cell lines, xenografts, and in a retrospective lymph node negative breast carcinoma population. The lymph node negative breast carcinoma study was comprised of 40 patients: 19 with nonrecurrent and 21 with recurrent disease. The 40 patients were matched according to age, cathepsin D, tumor size, percent S-phase, DNA ploidy, steroid receptor status, and tumor grade. Nm23-H1 protein levels in cell lines and xenografts were analyzed quantitatively using Western blot analyses and semiquantitatively in tissue sections using immunocytochemistry. Immunocytochemical analysis of lymph node negative breast tumors was graded as the percent of tumor staining positive for nm23 and the intensity of staining. The metastatic potentials of the cell lines and xenografts were assessed as the ability to form metastatic lesions in nude mice. In the lymph node negative breast carcinoma patients, the metastatic potential was characterized as the incidence of breast carcinoma recurrence. RESULTS: The MCF-7 cell line expressed four- and tenfold higher levels of nm23-H1 than the highly metastatic MDA-MB-435 and MDA-MB-231 cells, respectively. Among the xenografts and cell lines, there was an inverse correlation between nm23-H1 expression and metastatic potential in athymic nude mice (correlation coefficient [R] = -0.51). The differences between the levels of nm23-H1 among the metastatic and nonmetastatic cell lines and xenografts were not statistically significant. Statistical analyses indicated that neither the intensity nor the percent of tumor staining positive for nm23 expression was correlated to the recurrence of breast carcinoma in the lymph node negative patient population that had been matched for other clinical prognostic markers. CONCLUSIONS: There was an inverse correlation (R = 0.51) between the levels of nm23-H1 expression in cell lines and xenografts and the metastatic potential in nude mice. In the retrospective lymph node negative breast carcinoma population, no clear association was demonstrated between the expression of nm23 and breast carcinoma recurrence. This observation suggests the nm23 expression does not predict outcome in lymph node negative breast carcinoma patients.

Prevention of preneoplastic mammary lesion development by a novel vitamin D analogue, 1alpha-hydroxyvitamin D5.

BACKGROUND: The form of vitamin D (vitamin D3) in fortified milk and the provitamin D produced by the body undergo metabolic activation to a biologically active form, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This compound can induce cell differentiation and can prevent proliferation of cancer cells. However, because 1alpha,25(OH)2D3 is hypercalcemic (effective in increasing serum calcium level), it is not suitable for use in cancer prevention or cancer therapy trials. PURPOSE: We synthesized a vitamin D5 series analogue, 1alpha-hydroxy, 24-ethyl-cholecalciferol, or 1alpha-hydroxyvitamin D5 [1alpha(OH)D5], and evaluated its chemopreventive activity in carcinogen-treated mammary glands in organ culture experiments. METHODS: The analogue 1alpha(OH)D5 was synthesized from sitosterol acetate and was characterized by nuclear magnetic resonance. Its purity was evaluated by high-pressure liquid chromatography. The calcemic activities of vitamin D3 and D5 analogues were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of BALB/c mice were placed in organ culture and treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic lesions. Vitamin D analogues were added to the culture medium at four different concentrations, and formation of mammary lesions was evaluated. The effects of 1alpha(OH)D5 and 1alpha,25(OH)2D3 on the expression of vitamin D receptors (VDRs) and transforming growth factor-beta1 (TGF-beta1) were studied by immunohistochemistry. Statistical significance was determined by the chi-squared test. All reported P values were two-sided. RESULTS: 1alpha,25(OH)2D3 was fourfold more calcemic than 1alpha(OH)D5 at a dose of 0.042 microg/kg per day in rats. Both 1alpha,25(OH)2D3 and 1alpha(OH)D5 inhibited the development of DMBA-induced preneoplastic lesions in mouse mammary glands compared with untreated glands. The effect of the vitamin D3 analogue was observed at a much lower concentration (0.01 microM). Treatment with 1alpha(OH)D5 resulted in a dose-related (0.01-10.0 microM) inhibition without any toxicity, whereas the vitamin D3 analogue was highly potent but toxic at concentrations of 1.0 microM or higher. Normal mouse mammary glands poorly express VDR and TGF-beta1; incubation with 1alpha(OH)D5 or 1alpha,25(OH)2D3 dramatically induced their expression. CONCLUSIONS: This is the first report showing the possibility of chemoprevention by a vitamin D5 series compound. We conclude that 1alpha(OH)D5 is less calcemic than 1alpha,25(OH)2D3. It is nontoxic at a wide range of concentrations, but it is potent in inhibiting the development of preneoplastic lesions in mammary glands in organ culture. In addition, we show for the first time the induction of TGF-beta1 in normal mammary tissues by a chemopreventive agent. IMPLICATIONS: 1alpha(OH)D5 is a good candidate for in vivo chemoprevention studies. It may mediate its action by inducing expression of VDR and of TGF-beta1, as is seen in other systems.

Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii.

Triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii Hook f. (Celastraceae), has been shown to demonstrate potent antileukemic activity in rodent models at remarkably low treatment doses. A variety of other physiological responses are known to be mediated by this compound, including immunosuppressive and antifertility effects. We currently report that triptolide was not mutagenic toward Salmonella typhimurium strain TM677, either in the presence or absence of a metabolic activating system. Relatively potent but non-specific cytotoxicity was observed with a panel of cultured mammalian cell lines, and modest antitumor activity was observed when an i.p. dose of 25 microg was administered three times weekly to athymic mice carrying human breast tumors. Treatment regimens involving higher doses of triptolide (e.g. 50 microg/mouse three times weekly) were lethal.

Overexpression of mutant p53 and c-erbB-2 proteins and breast tumour take in mice.

We established a panel of 17 xenografts from primary human breast carcinomas. We examined which characteristics of the original tumours and the xenografts facilitate growth in animals. Tumours expressing medium or strong immunoreactivity for p53 protein had significantly (P < 0.05) higher incidence (92%) of in vivo tumour take than those showing weak or negative immunoreactivity (9.1%). No such association was observed between either c-erbB-2 or epidermal growth factor receptor (EGFR) expression in the original tumours and their in vivo tumour take. Following subcutaneous (s.c.) transplantation of original breast tumours or established xenografts, 7/17 tumours showed metastatic disease spread to distant sites (mainly lungs). This study suggests that selective growth of highly aggressive tumours occurs during in vivo propagation of malignant tumours, and these tumours will be of particular interest in evaluating various chemotherapeutic agents for breast cancer management.

A cell line derived from a clinically benign phyllodes tumor: characterization and implications.

Phyllodes are uncommon tumors of the breast. Improved understanding of their behavior is hampered by the paucity of good laboratory models. We have developed two cell lines, by both xenograft and direct cell culture, derived from a histologically benign phyllodes tumor. Both cell lines have the same characteristics and growth kinetics. They grow as monolayers of spindle-shaped cells, with surface markers consistent with a mesenchymal origin. They do not express either estrogen or progesterone receptors. The cells have a relatively short doubling time of just over 1.5 days, and show a stimulatory effect with the addition of insulin. Karyotype analysis reveals the absence of one X chromosome.

Regulation of 17 beta-hydroxysteroid dehydrogenase in a newly-established human breast carcinoma cell line.

UISO-BCA-1 human breast carcinoma cell lines, established and characterized in our own laboratory, were used to study both oxidative and reductive pathways of 17 beta-hydroxysteroid dehydrogenase (17 beta-OH-SDH). This enzyme has been suggested to catalyze conversion of both estrone to estradiol and estradiol to estrone. In order to determine the natural preferred enzymic pathway, the enzymic activity was assayed in intact cell monolayers. In these cells, reduction of estrone to estradiol was 7-fold higher than oxidation of estradiol to estrone. For the reductive pathway, the apparent Michaelis-Menten (Km) was 5.5 microM, and for the oxidative pathway, it was 14.3 microM. The enzymic conversion of estrone to estradiol was enhanced by 72 h treatment with estrone, estradiol and R5020, dehydroepiandrosterone, or dehydroepiandrosterone sulfate. On the other hand, oxidation of estradiol to estrone was stimulated by estradiol+R5020, but inhibited by estrone treatment. The results of the kinetic study, and regulation by various steroids in the present study, indicate that oxidation of estradiol or reduction of estrone is probably mediated via different forms of 17 beta-OH-SDH.

Growth and metastasis of human breast carcinomas with Matrigel in athymic mice.

Immunodeficient athymic mice with human tumor xenografts provide an important in vivo experimental model for cancer research. However, only a limited number of tumor types grow in these animals. For human breast carcinomas, the incidence of tumor-take is 6-15%. Recently, increased incidence of xenograft development in mice has been reported for various human tumors when the tumors were coinjected with Matrigel. We studied the development of human breast carcinoma xenografts in athymic mice with and without coinjection of Matrigel. Tumors developed in only 7.3% of enzyme-dispersed tumors injected subcutaneously in saline solution alone. None of these tumors metastasized to distant sites. On the other hand, 50% of enzyme-dispersed tumors coinjected with Matrigel developed xenografts; four out of five of these tumors metastasized to distant sites. Our data from the recent study suggest that, in athymic mice, Matrigel not only enhanced breast tumor growth but also facilitated tumor metastasis.

Steroid receptors in breast cancer patients. Influence of obesity and age at diagnosis.

The influence of host age on estrogen (ER) and progesterone (PR) receptor status was studied in 603 tumors obtained from women with confirmed diagnosis of breast carcinoma. Both ER and PR analysis were performed in our own laboratory using standard techniques. Tumors were classified as positive if minimum receptor contents were greater than 10 fmol/mg cytosol protein and if dissociation constants were 1-9 x 10(-10) M or lower. Data from our study indicate that the incidence of receptor negative (ER-PR-) tumors was higher in women from 21 to 40 years of age than in women from 41 to 60 years of age. In women over 60 years of age, the incidence of ER+PR+ tumors was higher than in women under 40 years of age. Interestingly, women from 51 to 60 years of age had a significantly lower incidence (P less than 0.06, 0.0001) of ER+PR+ but higher incidence (P less than 0.01) of ER-PR- tumors than women 41-50 or greater than 60 years of age. Analysis of steroid receptor distribution in relation to host age and obesity showed a definite tendency: in obese women over 60 years of age, frequency of ER+PR+ was significantly greater than in non-obese women of similar age groups. This altered ER and PR distribution in tumors is probably a result of difference in the hormonal milieu associated with host menopausal status and obesity.

Human breast carcinoma cell lines: ultrastructural, genotypic, and immunocytochemical characterization.

Two new breast carcinoma cell lines, designated as UISO-BCA-1 and UISO-BCA-2, have been established from pleural effusions of postmenopausal women. Both cell lines show properties of mammary epithelial cells, such as positive immunoreactivity to cytokeratins and human milk fat globulin, presence of desmosomal junctions, numerous microvilli, intracytoplasmic duct-like vacuoles and tonofilaments. UISO-BCA-1 and UISO-BCA-2 cells differ from each other with respect to cellular morphology, ultramicroscopic details, immunoreactivity to Her-neu oncogene protein, chromosomal mode and in vivo and in vitro growth rates. UISO-BCA-1 cells are well-differentiated (as evident from their morphology and ultrastructural details) and hyperploid (42-114 chromosomes). In vitro, UISO-BCA-1 cells are fast growing, with a population doubling time of 31.2 +/- 9.6 hrs (n = 4), and are tumorigenic (100%) in athymic nude mice. In contrast, UISO-BCA-2 cells are poorly differentiated, but are also hyperploid, with 54-64 chromosomes. UISO-BCA-2 cells are slow growing in vitro (population doubling time: 56.0 +/- 5.0 hrs [n = 4]) and have limited tumorigenic potency (20-40%). Both these cell lines are estrogen and progesterone receptor (less than 10 fmol/mg protein) negative.