Effects of social housing condition on chemotherapeutic efficacy in a Shionogi carcinoma (SC115) mouse tumor model: influences of temporal factors, tumor size, and tumor growth rate.

OBJECTIVE: The objective of this study was to investigate 1) whether social housing condition, tumor size, and tumor growth rate alter responses to chemotherapy and 2) whether the timing of tumor cell injection or chemotherapy initiation (relative to housing condition formation) influences tumor growth rate or the efficacy of chemotherapy. METHODS: Mice were reared individually (I) or in groups (G). In experiment 1, mice were rehoused (IG or GI) or left in group housing (GG) immediately after tumor cell injection. In experiment 2, housing conditions (II, IG, GG, or GI) were formed when tumors weighed 1 g. Chemotherapy (adriamycin 4 mg/kg and cyclophosphamide 61.5 mg/kg IP) and exposure to acute novelty stress (15 min/d, 5 d/wk) were initiated 1 day after housing condition formation. RESULTS: If chemotherapy was initiated when the tumor burden was undetectable (experiment 1), housing condition did not alter tumor response to chemotherapy, although IG mice lost the most weight and overall had the lowest probability of survival. If chemotherapy was initiated when tumors weighed 1 g (experiment 2), both tumor and host responses to chemotherapy were poorest for IG mice. Timing of tumor cell injection relative to housing condition formation also differentially influenced the rate of tumor growth in mice treated with the drug vehicle; in experiment 1, tumor growth rate was faster in GI and GG mice than in IG mice, whereas in experiment 2, the rate of tumor growth was faster in II mice than in GG and IG mice. CONCLUSIONS: Altering the temporal relationships among social housing condition formation, tumor cell injection, and chemotherapy initiation differentially influences the rate of tumor growth and the efficacy of chemotherapy. Effects of housing condition are independent of tumor growth rate at chemotherapy initiation and, in terms of host responses, independent of tumor burden.

Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue.

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted, myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM), alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18, keratin 19, EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors.

Effect of social housing condition on heat shock protein (HSP) expression in the Shionogi mouse mammary carcinoma (SC115).

Our previous studies have shown that social housing conditions can significantly alter the growth rate of the Shionogi mouse mammary carcinoma (SC115). The present study extended our investigations to the molecular level by examining stressor effects on the expression of a group of stress-responsive proteins, the heat shock proteins (HSPs). We hypothesized that HSP expression in SC115 cells may be altered by (a) different social housing conditions in vivo and (b) steroid hormone and growth factor exposure in vitro. Mice were reared in groups (G) or as individuals (I). Immediately following tumor cell injection, mice were rehoused from group to individual (GI), from individual to group (IG), or they remained in groups (GG). Tumor tissue was resected at 0.8 g or 3.0 g, as evidence suggests that tumor size affects HSP expression, which in turn affects proliferation. The data demonstrate that expression of HSP25, 70, and 90 was increased in tumors from mice in the IG compared to GG and GI mice, at both tumor weights examined. In addition, in IG mice, HSP90 expression was greater in 0.8 g compared to 3.0 g tumors. Under controlled culture conditions, hormones known to stimulate SC115 growth both in vivo and in vitro altered HSP expression. Physiological levels of dihydrotestosterone (DHT) and pharmacological levels of hydrocortisone (HC) upregulated expression of HSP25, whereas physiological levels of beta-estradiol (E2) upregulated expression of HSP90. These data are the first to demonstrate that a psychosocial stressor, a change in social housing condition, can induce differential HSP expression. Further, these data show that hormones that regulate SC115 tumor growth, also alter HSP expression.

Psychosocial stressors and mammary tumor growth: an animal model.

Stressful life events and the ability to cope with stress may play a role in the progression of breast cancer; however, the complex relationship between stressors and tumor growth is difficult to investigate in humans. Our studies have utilized the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice to investigate the effects of social housing condition on tumor growth rates and responses to chemotherapy. We demonstrate that, depending on social housing condition, mammary tumor growth and response to chemotherapy can both increase and decrease. We have examined the possible role(s) of 1) psychosocial variables, 2) testosterone and corticosterone, hormones altered by stress and known to stimulate SC115 cells in vivo and in vitro, 3) NK cells, one of the body's first lines of defense against tumor cells, 4) stress proteins, in mediating the differential tumor growth rates observed in our model. This review discusses the investigations we have undertaken to elucidate the mechanisms through which a psychosocial stressor, social housing condition, can alter tumor growth rate.

Interactive effects of psychosocial stressors and gender on mouse mammary tumor growth.

We have previously demonstrated that social housing condition significantly affects the growth rate of the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice. The present study examined the effects of social housing condition and acute daily exposure to a novel environment on the growth rate of an androgen-independent variant of the AR SC115 carcinoma, designated SC115V, in male and female mice. Immediately following tumor cell injection, male and female mice that were reared as individuals (I) or in groups (G) of the same sex were rehoused either from individual to same-sex groups (IG) or from group to individual (GI), or remained in their group housing condition (GG). Approximately half the mice in each housing condition were subjected to acute daily exposure to novel environments (novelty stress), a treatment shown previously to increase the significant difference in tumor growth rates between male mice in the IG and GI housing conditions. The remaining mice were left undisturbed (no novelty stress). In the presence of acute daily novelty stress, the growth rate of the SC115V tumor was significantly increased in GI compared to IG males. However, no significant differences in SC115V tumor growth rates among nonstressed GI, IG, or GG males were observed. For females, in contrast to males, acute daily novelty stress significantly decreased tumor growth in GI compared to IG mice, whereas under nonstressed conditions, tumor growth rate was significantly increased in GI compared to IG females. Neither housing condition nor novelty stress altered estrous cyclicity, nor did the stage of the estrous cycle at the time of tumor cell injection influence tumor growth rates. These findings suggest that social housing condition and novelty stress may interact to produce differential effects on the growth rate of the SC115V tumor in male and female mice.

Correlation of CD44 expression with proliferative activity of normal human breast epithelial cells in culture.

A number of studies have shown that certain variant isoforms of CD44 are overexpressed in human breast cancer, suggesting their use as indicators of the presence of malignant cells. We now show that CD44 isoform mRNA and protein expression is upregulated in normal human breast epithelial cells (HBEC) when these cells are stimulated to proliferate in culture. Reverse transcription-PCR analysis of cultured normal HBEC revealed complex patterns of CD44 mRNA expression that were indistinguishable from patterns previously shown to be characteristic of tissue samples containing malignant HBEC. CD44v6-expressing cells were identified in cultures generated from FACS-purified populations of either normal luminal (CALLA-MUC-1+) or myoepithelial (CALLA+MUC-1-) cells, even though immunohistochemical analysis of normal breast tissue sections confirmed CD44v6 expression to be limited to the myoepithelium in vivo. Increased expression of both CD44v mRNA and protein in cultured populations of normal HBEC was shown to correlate positively with the proportion of cells that were proliferating (Ki-67+) independent of cell density. These results indicate that activation of CD44 variant isoform expression in HBEC occurs as a normal response to factors that stimulate their proliferation and suggests caution in the use of this marker to identify malignant cells.

Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast.

The developmental relationships between the different mammary epithelial cell lineages in the human mammary gland are not well defined. To characterize human breast epithelial cells (HBEC) with progenitor activity, we used flow cytometry and single cell sorting to analyze the distribution of cellular phenotypes in primary cultures of reduction mammoplasties and their associated ability to generate colonies in 2-dimensional (D) and 3-D (collagen gel) culture systems. This approach allowed two distinct types of HBEC progenitor populations to be distinguished on the basis of their differential expression of the MUC-1 glycoprotein, CALLA/CD10 and epithelial-specific antigen (ESA). The first type of progenitor, which is enriched in the MUC-1+/CAL-LA-/ESA+ subpopulation, generated colonies of tightly arranged cells in 2-D cultures and small alveolar-like colonies with a central lumen when cultured in a collagen matrix. The cells produced in the colonies and derived from these MUC-1+/CALLA-/ESA+ progenitors were found to express typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, which is present in the MUC-1-to +/-/CALLA +/- to +/ESA+ subpopulation, generated mixed colonies of both luminal and myoepithelial cells when seeded in 2-D and 3-D cultures. In 2-D cultures, the centrally located cells exhibited a luminal morphology and expressed ESA, but were heterogeneous in their expression of MUC-1. Radiating from the periphery of these ESA+ HBEC were highly refractile ESA- teardrop-shaped myoepithelial-like cells. When cultured in a collagen matrix, these bipotent progenitors generated large branched colonies composed of a heterogeneous population of cells, with some of the progeny cells expressing luminal epitopes (keratin 8/18, keratin 19 and MUC-1) and others expressing myoepithelial epitopes (keratin 14 and CD44v6). A third type of progenitor, which became apparent is passaged HBEC cultures and was enriched in the MUC-1-/CALLA+/ESA- subpopulation, was found to generate colonies of cells with an exclusively myoepithelial phenotype. These results provide definitive evidence for the existence of multilineage HBEC progenitors in normal adult human mammary tissue. The phenotypic profile of these cells suggest that these multilineage progenitors are a relatively undifferentiated cell since they express low levels of MUC-1 and that they have a luminal location within the mammary epithelium since they are ESA+. Furthermore, we suggest that the MUC-1+/CALLA-/ESA+ and the MUC-1- to +/-/CALLA +/- to +/ESA+ progenitors we have identified and characterized are candidate in vivo alveolar and ductal progenitors, respectively.

Isolation and analysis of different subpopulations of normal human breast epithelial cells after their infection with a retroviral vector encoding a cell surface marker.

The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (< 9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.

Effects of social housing condition on the response of the Shionogi mouse mammary carcinoma (SC115) to chemotherapy.

In the present study, we demonstrate that social housing conditions significantly alter the response of the transplantable androgen-responsive Shionogi mouse mammary tumor (SC115) to chemotherapy. Mice were reared either in groups (G) or as individuals (I). Immediately following tumor cell or vehicle injection, mice were rehoused from group to individual (GI) or from individual to group (IG) conditions. A combination of Adriamycin (4 mg/kg) and cyclophosphamide (61.5 mg/kg), in a series of three i.p. injections 7 days apart, was initiated when mean tumor weights of mice within a housing condition (GI or IG) reached 1 g. Survival probability was significantly greater in mice in the IG housing condition compared to those in the GI housing condition (47% versus 19%, respectively). Additionally, the median survival time following the initiation of chemotherapy was greater for mice in the IG than for mice in the GI condition (24.5 days versus 15.0 days, respectively). These findings suggest that a psychosocial stressor, social housing condition, can significantly influence chemotherapeutic efficacy.

Effects of social housing condition and behavior on growth of the Shionogi mouse mammary carcinoma.

We have demonstrated marked effects of social housing condition on the growth rate of the androgen-responsive Shionogi mouse mammary carcinoma. The present study investigated the possible role of psychosocial variables in modulating the differential tumor growth rates observed. Male DD/S mice were reared individually housed (I) or in groups (G) of three or five siblings or nonsiblings. Following tumor cell injection, mice either remained in their rearing conditions (II, GG) or were rehoused (IG, GI). Effects of group size, sibling relationship, dominance status, change vs. no change in housing condition, and direction of change (individual to group or group to individual) were examined. Home cage behaviors were monitored both prior to and following tumor cell injection and rehousing. Overall, mice in the GI conditions showed faster tumor growth rates than mice in the IG conditions. Mice in the II and GG conditions showed intermediate tumor growth rates. Differences in group size and sibling relationship prior to and following tumor cell injection and rehousing had no significant influence on tumor growth rates. However, both change in housing condition and direction of change following tumor cell injection/rehousing were significant variables in modulating differential tumor growth rates. Dominance status differentially influenced tumor growth depending on whether mice experienced a change in housing; in the IG conditions, dominant mice showed faster tumor growth whereas in the GG conditions, dominant mice showed slower tumor growth than subordinate mice. Increased fighting among mice in IG compared to mice in GG conditions may play a role in modulating differential tumor growth rates.

The effects of cyclosporin A, tamoxifen, and medroxyprogesterone acetate on the enhancement of adriamycin cytotoxicity in primary cultures of human breast epithelial cells.

Adriamycin (Adr), the single most active agent used in the treatment of breast cancer, may become ineffective as treatment progresses due to the development of multidrug resistant (MDR) tumors. A major mechanism associated with MDR is increased P-glycoprotein (Pgp) expression. This study examined the abilities of the anti-estrogen tamoxifen (TAM) and the progestin medroxyprogesterone acetate (MPA) as well as cyclosporin A (CsA), a known resistance modifier, to enhance the cytotoxic effects of Adr on human breast epithelial cells (HBEC) in primary culture. Pgp and estrogen receptor (ER) expression were determined in each of the cultures by immunocytochemical assays using the monoclonal antibodies C219 and H222 Sp gamma, respectively. The Adr-sensitive, Pgp-, ER+ MCF-7 cell line and the Adr-resistant, Pgp+, ER- MCF7-AdrR cell line were used as controls. Primary cultures were categorized as HBEC from tissues with or without previous chemotherapy. Pgp was detected in 1 of the 15 cell cultures from tissues without previous chemotherapy and in 5 of the 6 cell cultures from tissues previously exposed to chemotherapy. Incubation with either CsA or MPA plus Adr enhanced Adr toxicity in Pgp+ but not Pgp- cell cultures, whereas TAM had no effect on the sensitivity of any of the cultures. Of the 21 primary cultures of HBEC, 3 were ER+. There was no correlation between the enhancement of Adr cytotoxicity and ER status. The data suggest that MPA as well as CsA may be useful as modifying agents in overcoming Pgp-associated multidrug resistance.

Selective growth of freshly isolated human breast epithelial cells cultured at low concentrations in the presence or absence of bone marrow cells.

In this study, we show that conditions previously found to promote the selective growth of human breast epithelial cells (HBEC) in serum-free primary cultures established from normal or malignant tissue can be extended to cultures initiated at low seeding densities (< 5000 cells/cm2). The epithelial nature of the cells produced was documented by their positive staining with antibodies specific for keratins 8, 14, and 18, and 2 antibodies that recognize epithelial-specific antigens (Ber-EP4 and HB8630). HBEC growth was not affected, either positively or negatively, by the use of a medium containing a combination of fetal calf and horse serum, which promotes the growth of many types of stromal cells and associated hematopoietic precursors, or by the inclusion in the initial cell suspension of marrow cells at HBEC to marrow cell ratios typical of bone marrow samples from patients with metastatic breast cancer. The presence of fibroblast feeders from a variety of sources enhanced the growth of HBEC to different degrees. In cultures initiated with low numbers of cells obtained from samples of breast carcinoma, HBEC growth was generally reduced by comparison to cultures of normal HBEC. With the detection methods used, it was not possible to determine the extent to which this decreased growth was due to a reduced frequency of malignant HBEC with in vitro precursor activity, or the presence of reduced numbers of residual normal HBEC precursors, or both. However, preliminary data indicate that this approach also allows the detection of some breast carcinoma cells with proliferative ability that are present in the marrow or pleural effusions of some breast cancer patients. These studies demonstrate the feasibility of detecting normal and malignant HBEC with growth potential when these are cultured at low density and/or as rare contaminants of marrow cell suspensions, and provide a starting point for their further characterization.

Role of natural killer cells in psychosocial stressor-induced changes in mouse mammary tumor growth.

We have shown that social housing conditions affect the growth rate of the androgen-responsive Shionogi mouse mammary tumor (SC115) and differentially stimulate splenic natural killer (NK) cell activity. Mice reared in a social group and then singly housed (GI) following tumor cell injection have increased tumor growth rates and increased NK cell activity, whereas mice reared individually and then group housed following tumor cell injection have decreased tumor growth rates and decreased NK cell activity compared to that in mice remaining in their rearing group. The present study was undertaken to determine whether NK cells are involved in mediating the effects of social housing conditions on SC115 tumor growth rates. We demonstrate that the presence of the SC115 tumor significantly stimulates the activity of NK cells at the tumor site in the first 7 days after tumor cell injection, and that, consistent with the data on splenic NK cells, mice of the GI group (largest tumors) have significantly greater levels of NK cell activity than mice reared individually and then group housed (smallest tumors). We further demonstrate that in mice of the GI group, in vivo stimulation of NK cell activity by polyinosinic:polyCMP correlates with a corresponding increase in tumor growth rate. These findings suggest that NK cell activity may play a role in mediating the increased tumor growth rate observed in mice of the GI housing condition.

Lack of effect of hematopoietic growth factors on human breast epithelial cell growth in serum-free primary culture.

A number of recombinant cytokines believed to regulate normal hematopoiesis are now being used in cancer treatment protocols to reduce the myelosuppressive toxicity of intensive chemoradiotherapy regimens. It is widely assumed that such cytokines are relatively specific for hematopoietic cells, although some cell lines derived from a variety of non-hematopoietic human tumors can respond to some of these factors. However, relatively little is known about their ability to stimulate (or inhibit) the proliferation of freshly isolated normal or malignant non-hematopoietic cells. We have used a serum-free culture medium that selectively supports the growth of human breast epithelial cells (HBEC) obtained directly from normal or malignant tissue samples to evaluate potential stimulatory or inhibitory effects of eight cytokines: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Steel factor, interleukin-2, interleukin-3, interleukin-6, transforming growth factor-beta and macrophage inflammatory protein-1 alpha, on these cells cultured both in the presence of epidermal growth factor, a potent stimulator of HBEC growth, and in its absence. HBEC growth was assessed after 7 and 14 days using the tetrazolium-dye reduction assay. Potential effects on the well studied MCF-7 breast cancer cell line, cultured under the same conditions, were also investigated. None of the cytokines (which were tested over a wide range of concentrations) had any modulating effect on the growth of normal or malignant HBEC under the conditions used with the exception of transforming growth factor-beta, which was consistently and significantly inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of steroid hormones and opioid peptides on the growth of androgen-responsive Shionogi carcinoma (SC115) cells in primary culture.

We investigated the effects of dihydrotestosterone (DHT), hydrocortisone (HC), basic fibroblast growth factor (bFGF), and opioid peptides on the growth of cells from the androgen-responsive Shionogi mouse mammary carcinoma (SC115) in primary culture. Androgen-responsive SC115 tumor cells were stimulated to grow in response to DHT, HC, and bFGF in a dose-responsive manner in both serum-containing and serum-free media. Moreover, anti-bFGF antibody had a marked inhibitory effect on DHT- and bFGF-induced growth. Three opioid agonists, beta-endorphin (beta-EP), cyclazocine, and morphine sulfate, markedly inhibited SC115 tumor cell growth at concentrations ranging from 10(-11) to 10(-7) M in serum-containing medium with or without DHT, HC, or bFGF, with the greatest inhibition occurring in medium with DHT. In serum-free medium, beta-EP had no inhibitory effects on cell growth. However, beta-EP at concentrations of 10(-9) M or greater significantly inhibited cell growth in serum-free medium containing DHT, HC, or bFGF, with the greatest inhibition again occurring in medium with DHT. Naloxone (10(-8) and 10(-6) M), an opioid receptor antagonist, blocked the inhibitory effects of beta-EP and morphine sulfate. These results suggest that SC115 tumor cells in primary culture are stimulated to grow in a dose-responsive manner by DHT, HC, or bFGF in both serum-containing and serum-free media. It appears that bFGF may mediate, at least partially, DHT-stimulated cell growth. In addition, the opioid peptide system may be involved in regulating endocrine control of growth of the androgen-responsive SC115 carcinoma. The dose-responsive inhibitory effects of opioids and their reversal by naloxone suggest that these effects may be mediated by opioid receptors.

Endocrine mediation of psychosocial stressor effects on mouse mammary tumor growth.

We have demonstrated that differential housing alters the growth rate of the androgen-responsive Shionogi mouse mammary carcinoma (SC115). In the present study we wished to determine if changes in plasma levels of hormones or a shift in the responsiveness of the tumor cells to hormones was responsible for the differential tumor growth rates observed. Plasma testosterone and corticosterone levels were assayed 24 h, 3 days and 1 week post tumor cell/vehicle injection. Also 3 weeks post injection androgen and glucocorticoid receptor binding capacity (Bmax) and binding affinity (Kd) and the in vitro responsiveness of tumor cells to dihydrotestosterone and hydrocortisone were measured. At 24 h post injection, plasma testosterone levels were significantly increased in mice with large tumors, but remained low in mice with small tumors. Plasma corticosterone levels were significantly elevated in mice with small tumors compared to those of mice with large tumors at all time points measured. Androgen and glucocorticoid receptor binding capacity and binding affinity of tumor cells did not differ among groups. Further, all groups tested had the ability to respond to dihydrotestosterone and hydrocortisone in vitro. These data indicate that an effect of housing condition on plasma levels of steroid hormones may, in part, mediate the differential tumor growth rates observed in this model.

Effects of estrogen, epidermal growth factor, and transforming growth factor-alpha on the growth of human breast epithelial cells in primary culture.

Since 17 beta-estradiol (E2)-stimulated growth in human breast cancer cell lines has been shown to be accompanied by increased production of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and their receptor, we investigated the effects of E2 and these growth factors on the growth of human breast epithelial cells (HBEC) in primary culture. HBEC from normal, benign, and malignant tissues were cultured in serum-free medium [DME:F12(1:1), 5 mg/ml BSA, 10 ng/ml cholera toxin, 0.5 micrograms/ml cortisol, 10 micrograms/ml insulin] in the presence and absence of E2, EGF, and TGF-alpha. Tritiated-thymidine ([3H]TdR) incorporation into DNA was used as a measure of cell growth. E2 did not stimulate growth of any of the cultures at all concentrations examined (10(-9) to 10(-6) M). In contrast, EGF ranging from 1 to 100 ng/ml consistently increased the growth of cells of all three breast tissue types in a dose-dependent manner. The EGF stimulation was inhibited by MAb 528, a monoclonal antibody against the EGF receptor. TGF-alpha was equally or more effective in stimulating proliferation, although its dose-response range was different than that of EGF. E2 and EGF together acted in a synergistic manner in 50% of the samples examined. These studies suggest that E2 can exert effects on HBEC growth via modulation of the cells' response to EGF.

In vitro screening of crude extracts and pure metabolites obtained from marine invertebrates for the treatment of breast cancer.

A total of 15 samples (crude extracts and pure secondary metabolites) obtained from marine invertebrates collected from the offshore waters of British Columbia, Papua New Guinea, and Sri Lanka have previously been shown to exert cytotoxic activity in the in vitro L1210 leukemic bioassay. We screened these metabolites for in vitro cytotoxic activity against the drug-sensitive breast-tumor cell lines MCF-7, T-47D, ZR-75-1, and MDA-MB-231; the multidrug-resistant and P-glycoprotein (Pgp)-positive breast-tumor cell lines MCF-7 Adr and MDA-A1r; and normal and malignant human breast epithelial cells (HBEC) in primary culture. Eight samples exhibited significant [drug concentration resulting in a 50% decrease in cell growth as compared with controls (ED50), less than 25 micrograms/ml] dose-dependent cytotoxicity against the drug-sensitive cell lines; the ED50 values were as low as 0.004 micrograms/ml. Five of the eight samples exhibited significant cytotoxicity against the multidrug-resistant cell lines; the ED50 values were as low as 0.0006 micrograms/ml. Incubation of MCF-7 Adr cells with varying concentrations of compounds in the presence of Adriamycin demonstrated that none of the compounds tested interfered with Pgp function. Results obtained using HBEC in primary culture showed a wide range of chemosensitivities for a given drug against tissue taken from different patients, demonstrating the uniqueness of the response of different individuals to chemotherapy.

Routine culturing of normal, dysplastic and malignant human mammary epithelial cells from small tissue samples.

We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely produce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression.