Cultures Of Mammary Cells Research Articles

Immunomodulation by soluble factors from tumor cells cultured in vivo in diffusion chambers.

The delayed-type hypersensitivity (DTH) response and lymphocyte-mediated angiogenesis were determined in mice bearing in vivo cultures of mammary tumor cells in diffusion chambers (DCs). Soluble tumor products which diffuse from the DCs were able to stimulate the immune system for both the DTH reaction and angiogenic activity by spleen cells.

Levels of viral glycoprotein reflect enhanced effectiveness of combined drug treatments

A chemosensitivity assay with small replicate Mm5mt/cl C3H mammary tumor cell cultures was developed to determine whether charges in viral antigen expression and release into culture fluids could be utilized as an in vitro measure of single and combined drug effect. The measurement of drug concentrations required for identical 50% reductions in viral antigen levels (ED 50s) allowed the dosage-dependence of the same drug to be compared alone and in combination drug treatments. The 52,000 MW viral envelope glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) was measured in culture fluids of control and drug-treated cultures while cell density was simultaneously determined by cell staining and OD 664 mu determination. While extracellular gp52 levels and cell density increased over 72 hours in control cultures, dose-dependent declines in both parameters provided dual measures of effect for combination CAF treatment [cyclophosphamide: doxorubicin (Adriamycin): 5-fluorouracil], combination CMF treatment [cyclophosphamide: methotrexate: 5-fluorouracil] and single component treatments of these combinations. Comparison of ED 50s for gp52 reduction revealed that each drug's ED 50 concentration was lower in combination treatment than it was in single treatment. In combined drug treatments, the ED 50 of doxorubicin was lowered from 5.17 μM to 0.36 μM, that of 5-fluorouracil was lowered from 0.69 μM to 0.22 μM and that of methotrexate was lowered from 15 nM to 1.65 nM. Reductions of ED 50 in combined drug treatments argued for drug synergisms in these combinations. When drug concentrations, rather than gp52 reductions, were fixed at identical levels in single and combined treatments, additional declines in gp52 levels reflected the enhanced effects of CAF and CMF combinations over single drug treatments. When the anti-estrogen, tamoxifen, was added to CAF and CMF treatments, further declines in gp52 levels (below CAF and CMF levels) reflected the fact that these hormone-augmented treatments were more effective than either hormone or combination chemotherapy alone. These results indicate that decreases in extracellular levels of MMTV gp52 can be utilized in this in vitro assay an an alternative measure of chemotherapeutic effect for evaluating the synergistic of antagonistic nature of drug or hormone interactions and as a tool for in vitro optimization of combined drug effect.

Normal breast epithelial cells produce interleukins 6 and 8 together with tumor-necrosis factor: defective IL6 expression in mammary carcinoma.

Virtually pure primary cultures of normal mammary epithelial cells (MEC) obtained from healthy women were shown to release interleukin 6 and 8 (IL6, IL8) and to produce a nonsecreted form of tumor-necrosis factor (TNF). No interferon (IFN), whether alpha, beta, or gamma, or IL1-alpha or -beta could be detected. Analysis of cellular RNA confirmed these findings and showed that MEC also express IL6 receptor and TNF-alpha-related mRNAs. Epithelial cells were selectively stained by antibodies to IL6, IL8 and TNF-alpha both in primary cultures and in the normal mammary gland. Samples of human milk contained sizable amounts of IL6, IL8 and IFN-gamma; yet the liquid phase was consistently negative for other cytokines (i.e., TNF-alpha, IFN-alpha/-beta, IL1-alpha/-beta). Expression of IL6 (but not of IL8 and TNF-alpha) was abolished in ductal infiltrating carcinomas and greatly reduced in cultures of oncogene-transfected mammary cells, suggesting that alterations of IL6 expression are associated with pathogenesis in breast cancer.

Comparison of several methods to obtain bovine lactating epithelial mammary cells in culture : C. Claudon and F. Laurent. Laboratoire des Sciences et Techniques des Productions Animales E.N.S.A.I.A. (I.N.P.L.), BP 172,54505 Vandoeuvre, France

Comparison of several methods to obtain bovine lactating epithelial mammary cells in culture : C. Claudon and F. Laurent. Laboratoire des Sciences et Techniques des Productions Animales E.N.S.A.I.A. (I.N.P.L.), BP 172,54505 Vandoeuvre, France

Steroidal inhibitors as chemical probes of the active site of aromatase

Androstenedione analogs containing 7α-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7α-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450 arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7α-(4′-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7α-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent K inact for 7α-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 × 10 −3 sec −1, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7α-(4′-iodo)phenylthio-1,4-androstadienedione, 7α-IPTADD, and the reconstituted aromatase system. Incubations with [ 125I]7α-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450 arom by gel elctrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450 arom. HPLC radiochromatographic analysis of the isolated cytochrome P50 arom confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P50 arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450 arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7α-IPTAPP binds covalently to the cytochrome P450 arom.

Effects of hyperthermia on intracellular calcium concentration and responses of cancerous mammary cells in culture.

Effects of hyperthermia on the intracellular calcium concentration (Cai) of an established mouse breast cancer cell line, MMT060562, were studied using fura-2 fluorescence microscopy and the whole-cell clamp technique. A sudden change of temperature from 37 to 45 degrees C induced a transient increase in the fluorescence ratio permeability of the cell membrane and inward current. Deletion of extracellular calcium abolished the fluorescence ratio response to the rise in temperature. Cai of some cells increased after hyperthermia treatment at 44-48 degrees C for 20 min, but the average increase of Cai was negligible. After hyperthermia treatment, spontaneous oscillation of Cai, chemical responses to ATP and bradykinin and the mechanically-induced spreading response diminished. However, the mechanically induced increase of Cai within the stimulated cell remained even after hyperthermia treatment. Suppression of the ATP-induced Cai response recovered to about half the original level within 12 h. Blockage of protein synthesis with cycloheximide (100 microM) had no effect on the recovery. The D-myo-inositol 1,4,5-triphosphate (IP3)-dependent increase of Cai remained intact even after hyperthermia treatment. It is concluded that hyperthermia treatment increases both the permeability of the cell membrane and Cai, but decreases the sensitivity of cells to ATP and bradykinin, presumably due to modification of the signal transduction mechanism.

Bovine Mammary Myoepithelial Cells. 1. Isolation, Culture, and Characterization

The objective of this study was to isolate, purify, culture, and characterize myoepithelial cells from bovine mammary glands. Myoepithelial cells were separated from other mammary and blood cells after collagenase digestion and centrifugation using metrizoate-ficoll gradients. Myoepithelial cells were identified by their characteristic morphology and cloned using selective detachment. They contained many densely packed myofilaments, very few cytoplasmic organelles, elongated surface projections, and a dense, irregularly shaped nuclei. Some cells were as large as 1.2mm in culture. Myoepithelial cells contained an extensive network of cytoskeletal proteins, including α-smooth muscle actin, α-actinin, and vimentin. When cultured, they tended to repel one another and never grew as closely associated cells. The myoepithelial nature of these cells was verified by showing that they contracted in response to oxytocin, bound oxytocin, and did not produce casein. Myoepithelial cells from fetal and lactating glands grew very well in culture. Active division of myoepithelial cells could be maintained for at least 3 mo, and cells could be serially subcultured at least seven times. The successful isolation and culture of bovine mammary myoepithelial cells make utilization of these cells possible in order to study their role in mammary growth and differentiation and milk ejection.

Correlation of hyaluronic acid accumulation and the growth of preneoplastic mammary cells in collagen: a longitudinal study.

Hyaluronic acid accumulation is characteristic of mammary tumor cells, and the amount that accumulates seems to correlate with the degree of malignancy of the producing cells. We have tested directly the relationship between hyaluronic acid accumulation and the replication rate of preneoplastic mammary cells in culture. We used nontumorigenic but immortal CL-S1 mouse mammary cells that were derived from a hyperplastic alveolar nodule. Using a collagen gel culture system, we found clear differences in the growth properties of cells before and after Passages 68 to 70. Late passage cells replicated earlier and faster than early passage cells in collagen and on plastic. The rate of cycling resembled that of tumorigenic mouse mammary cells during the first week of culture. Cells seeded at low densities cycled faster than those seeded at high densities during the second week in culture. Exogenous hyaluronic acid, at 10 to 1000 micrograms/ml, neither enhanced nor inhibited CL-S1 cell growth significantly in collagen, regardless of passage. However, by the third day in collagen, late passage cells produced 7 times more total glycosaminoglycans and 12 times more hyaluronic acid per cell than did early passage cells. Late passage cells also deposited 12 times more labeled hyaluronic acid in the matrix than did early passage cells, on a per-cell basis. After a decline in the deposition of hyaluronic acid in the extracellular matrix, growth ceased. The late passage cells did not grow in soft agar, indicating that they had not become neoplastic spontaneously during passage. However, their accelerated growth rate, coupled with the synthesis and secretion of large amounts of hyaluronic acid into the extracellular matrix, may characterize a distinct step in tumor progression in preneoplastic CL-S1 cells.

Parathyroid hormone‐related protein production by primary cultures of mammary epithelial cells

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult non-neoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/10(5) cells x 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)-beta, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.

Effects of the cimaterol (CL 263,780) on growth and cellular metabolisms in the mammary gland of rats

This study was conducted to examine the effects of cimaterol feeding on growth performances and mammary growth and differentiation. Attempts were also made to investigate the cellular metabolisms of protein and fat in the mammary gland at the various physiological stages (virgin, pregnancy, early lactation, and late lactation). Ninety-six female Sprague Dawley rats (approximately 52.3 g) were randomly assigned to three treatments: CON (feeding the diet without cimaterol), CIM I (feeding the diet with cimaterol [10 mg/kg] from weanling to parturition), CIM II (feeding the diet with cimaterol from breeding to parturition). Cimaterol improved ( P < 0.01) body weight gain and feed intake and reduced ( P < 0.01) feed efficiency. In the mammary gland, DNA, RNA, and protein content were increased, and lipid content was decreased, by feeding cimaterol. Also, functional activity (RNA/DNA) and size (protein/DNA) of mammary gland cells were increased by cimaterol. It was clear that the lipogenicactivity in the mammary gland was increased with lactogenesis, although cimaterol had no effect on lipogenic activity. But cimaterol was effective in increasing lipolytic activity. As a result of in vitro acinar mammary cell culture, cimaterol showed a direct effect on increasing protein synthetic activity ( P < 0.01). It was observed through this study that cimaterol functions not only on energy repartitioning but also on growth and mammary development and differentiation in rats.

A T.E.M. study of mouse mammary epithelial cell secretory differentiation cultured on collagen and Matrigel

It has been an interest of our lab to develop a mammary epethelial cell culture system that faithfully duplicates thein vivocondition of the lactating gland. Since the introduction of collagen as a matrix on which cells are cultivated other E.C.M. type matrices have been made available and are used in many cell culture techniques. We have previously demonstrated that cells cultured on collagen and Matrigel do not differentiate as they doin vivo. It seems that these cultures often produce cells that show a disruption in the secretory process. The appearance of large ribosomal studded vesicles, that specifically label with antibody to casein, suggest an interruption of both protein maturation and secretion at the E.R. to golgi transition. In this report we have examined cultures on collagen and Matrigel at relative high and low seeding densities and compared them to cells from thein vivocondition.

Estradiol Down-Regulates the Mannose-6-Phosphate/Insulin-Like Growth Factor-II Receptor Gene and Induces Cathepsin-D in Breast Cancer Cells: A Receptor Saturation Mechanism to Increase the Secretion of Lysosomal Proenzymes

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.

Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture

Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels.

Consequences of selenite supplementation on the growth and metabolism of cultures of canine mammary cells

Previous studies with cultures of canine mammary cells revealed differences in the degree of growth inhibition caused by selenite supplementation, with canine mammary tumor cell line 13 > 11 ⪢ non-neoplastic canine mammary cells. The present studies show this variation in growth retardation cannot be explained by selenium retention. Intracellular glutathione related inversely to the degree of growth inhibition resulting from the addition of selenite. Dimethyl selenide formation by S-9 preparations corresponded to the sensitivity of the culture to supplemental selenite. DL-buthionine-SR-sulfoximine, a specific inhibitor of glutathione biosynthesis, accentuated the growth inhibition and prevented the increase in intracellular glutathione caused by supplemental selenite. Treatment of canine mammary tumor cell line 13 cultures with DL-buthionine-SR-sulfoximine resulted in a persistent depletion of intracellular glutathione without affecting growth. Glutathione reductase activity, before and following selenite, was inversely related to the degree of growth inhibition, with canine mammary tumor cell line 13 > 11 > non-neoplastic canine mammary tumor cell line. Selenite addition increased the activity of γ-glutamylcysteine synthetase in canine mammary tumor cell line 11 and non-neoplastic canine mammary cells, but not in canine mammary tumor cell line 13 cells. The present data suggest the differences in the growth inhibition caused by selenite among these mammary cells is related to glutathione regulation and ultimately to selenium detoxification.

Influence of mammary cell differentiation on the expression of proteins encoded by endogenous BALB/c mouse mammary tumor virus genes

The interactions between differentiation-associated cellular events in the intact mammary gland or in cultured mammary cells and the post-transcriptional activity of the endogenous mouse mammary tumor virus (MMTV) loci were investigated. The transcriptional activities of the endogenous MMTV proviruses of the BALB/c mouse strain ( Mtv-6, Mtv-8 and Mtv-9) appear to be regulated differentially during pregnancy-induced mammary gland development (J.E. Knepper D. Medina and J.S. Butel J. Virol. 59, 518–521, 1986). Analysis of MMTV-specific proteins at various stages of mammary gland development (virgin, midpregnant, lactating, regressing) established the presence of steady-state levels of a 67,000- M r env precursor-type polypeptide at all physiological stages. However, processing to lower-molecular-weight env-specific proteins, including a predominant 50,000- M r species, was detected only with the transition to the functional mammary gland phenotype. The contributions of cell proliferation, cell-matrix interactions, and modulation of functional activity to the pattern of endogenous MMTV protein expression were investigated using a 3-dimensional collagen type I culture system. Growth and cell-matrix interactions (cell polarization, lumen formation) leading to formation of 3-dimensional duct-like structures were permissive for the synthesis and processing of MMTV-specific proteins; accumulation of high levels of the 50,000- M r env-specific polypeptide was associated with the onset of the fully functional mammary cell phenotype. Expression of MMTV-specific proteins was not due to amplification of a specific cell subpopulation. The potential of the full-length Mtv-8 and Mtv-9 proviruses to be transcribed, as indicated by their methylation status, was not dramatically different between differentiated and undifferentiated mammary cells in culture. This study indicates that MMTV transcriptional activity is reflected at the protein level in mammary tissue of BALB/c mice and that viral protein synthesis and processing may serve as important markers of different physiological stages of mammary epithelial cells. These observations also suggest a general approach to the examination of potential modulatory effects of cellular interactions (cell-cell, cell-matrix or both) known to be important in various differentiated epithelial cell systems for the expression of viral genes.

Catechol estrogen formation in MCF-7 cell culture and effects of bromoestrogen inhibitors

Agonist and antagonist activities have been reported for several catechol estrogens given exogenously. Since the metabolic clearance rate for catechol estrogens in the body is very rapid, catechol estrogens produced at other tissues will have minimal effect on breast tissue. Information of the extent of catechol estrogen formation within cells is critical in assessing the overall importance of these estrogen metabolites. Investigations of the conversion of estrogens to catechol estrogens were performed in the MCF-7 human mammary carcinoma cell culture system. Reverse-phase high-performance liquid chromatography (HPLC) analysis demonstrated that very little metabolism of estradiol occurs after 48 h, with only small amounts of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, and estriol being observed. The total amount of 2-hydroxyestrogen products formed from 1 μM estradiol was 406.2 pmol (SD = 60.9) per 3 × 10 7 cells in 48 h. Similar results were obtained using the simpler radiometric assay for estrogen 2-hydroxylase, which measures the release of 3H 2O from [2- 3H]estradiol. The effects of inhibitors of estrogen 2-hydroxylase were also examined in MCF-7 cells. 2-Bromoestradiol, 4-bromoestradiol, and 2,4-dibromoestradiol effectively block estrogen 2-hydroxylase in a dose-dependent manner in MCF-7 cultures, with ED 50 of approximately 1 μM for each inhibitor. Furthermore, these bromoestrogens bind poorly to estrogen receptors in MCF-7 cells and do not alter cell growth. Thus, in MCF-7 mammary cell cultures, metabolism of estradiol occurs to only a minor degree, and it is unlikely that the levels of catechol estrogens would reach physiologically relevant concentrations in the intact breast cancer cells. In addition, the bromoestrogens are effective inhibitors of estrogen 2-hydroxylase in cell culture systems, bind poorly to the estrogen receptor, and do not exhibit estrogenic effects at concentrations up to 1 μM.

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA > DEX > prednisolone > hydrocortisone > triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4–2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10 −8 MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.

Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types.

A medium is described that supports long-term growth in culture of human primary mammary tumor cells, of normal epithelial cells from mammoplasty, and of mammary tumor cell lines. Tumor cells are shown to be distinguishable from normal mammary epithelial cells by morphology, by growth requirements, and by two markers: preferential expression of the HMFG-2 epitope on tumor cells and preferential retention in tumor cell mitochondria of the lipophilic fluorescent dye rhodamine 123. Differential fluorescence of HMFG-2 fluorescein-conjugated antibodies can be used as a basis for separation of normal and tumor cells in a fluorescence-activated cell sorter, as can differential retention of rhodamine 123.

Radiosensitivity and PLDR in Primary Cultures of Human Normal and Malignant Mammary and Prostate Cells

Radiosensitivity and PLDR in Primary Cultures of Human Normal and Malignant Mammary and Prostate Cells

Levels of viral glycoprotein provide a dose-dependent measure of tumor cell inhibition

A chemosensitivity assay utilizing small replicate Mm5mt/c1 C3H mammary tumor cell cultures was developed to determine whether changes in viral antigen expression and release into culture fluids could be utilized as an in vitro measure of chemotherapeutic drug effect. The 52, 000 MW viral envelope glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) was measured in culture fluids of control and drug-treated cultures while cell density was simultaneously determined by cel staining and OD 664 mu determination. While extracellular gp52 levels and cell density both progressively increased over 72 hours for control cultures, treatments with doxorubicin resulted in dose-dependent declines in both parameters at 24, 48, and 72 hours. Comparison of doxorubicin dosages for 50% reduction (ED 50s) in both parameters (0.68uM and 1.1uM) revealed a similar coordinate reduction in both cell density and MMTVgp52. When gp52 levels were further examined as a general measure of effect for a broad spectrum of 6 drugs with differing mechanisms of action, coordinate declines in cell density and MMTVgp52 provided a time and dose-dependent dual measure of effect for each of the drugs tested. Coordinate declines resulted in the same following hierarchy of concentration-dependent drug potency: methotrezate >62; 5-fluorouracil >62; doxorubicin >62; N [ phosphonacetyl- L aspartic acid] (PALA) >62; cis-platinum >62; cyclophosphamide. The dual measures of therapeutic effect afforded by this assay argue for its use as an in vitro measure of effect for optimizing drug treatments.