Showing papers on "Cancer cell published in 1983"

Hypomethylation distinguishes genes of some human cancers from their normal counterparts.

Abstract: It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture. The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients.

Inhibition of Human Cancer Cell Growth by 1,25-Dihydroxyvitamin D3 Metabolites

Abstract: Specific high-affinity 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors, which can undergo hormone-dependent activation and nuclear localization, have been demonstrated in a wide variety of established human cancer cell lines and surgically obtained human cancer tissues. 1,25-(OH)2D3 has been reported by some workers to stimulate cancer cell replication at low "physiological" concentrations and by ourselves and others to inhibit at higher concentrations. We report here that 1,25-(OH)2D3 had a biphasic effect on the replication of two distinct human cancer cell lines, i.e., the breast cancer T-47D and the malignant melanoma MM96, an effect analogous to that of estrogens on the breast cancer cell line MCF-7. These inhibitory effects were accompanied by marked morphological changes. Furthermore, two known metabolites of 1,25-(OH)2D3, i.e., 1,24,25-trihydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3, which compete for binding to the 1,25-(OH)2D3 receptor, did not stimulate but were almost equipotent with 1,25-(OH)2D3 in inhibiting the replication of both cell lines. The stimulatory but not the inhibitory effect of 1,25-(OH)2D3 was abolished by cortisone. These 1,25-dihydroxyvitamin D3 metabolites show promise for the inhibition of cancer growth, analogous to the effect of estrogens and antiestrogens in breast cancer but with potential application in a much wider range of human cancers.

Effects of tamoxifen on human breast cancer cell cycle kinetics: accumulation of cells in early G1 phase

Abstract: We have studied the effects of tamoxifen on the cell cycle kinetics of the endocrine-responsive MCF-7 human breast cancer cells. Tamoxifen inhibits proliferation of MCF-7 cells. The tritiated thymidine labeling index is markedly reduced by tamoxifen, indicating a reduction in the fraction of cells in S phase. Flow cytometry of mithramycin-stained cells reveals that cells accumulate in G1 phase, with a concomitant depletion of S- and G2-M-phase cells with tamoxifen. Mapping of G1-phase cells by morphology of prematurely condensed chromosomes demonstrated that tamoxifen-treated cells accumulate in early G1. These studies indicate that tamoxifen inhibits proliferation of MCF-7 human breast cancer cells by invoking a transition delay early in the G1 phase of the cell cycle.

Effect of Estradici on Human Breast Cancer Cells in Culture

Abstract: Conditions are described for growing and maintaining the estradici sensitivity of the human breast cancer cell line ZR75-1 both in monolayer and suspension cultures. Either new born calf or fetal calf serum can be used in the culture medium, but an effect of estradici on growth of the cells was only observed reproducibly if the serum was first treated with dextran-charcoal. Sulfatase treatment of the sera prior to dextrancharcoal treatment did not decrease cell growth in the absence of added estradiol, indicating that estrogen sulfates are unlikely to contribute to cell growth in dextran-charcoal-treated sera. In monolayer cultures, estradiol increased both the growth rate and final saturation density of the cells for each individual plating density tested in a dose-dependent manner with maxi mal stimulation occurring between 10~'°and 10~8 M estradiol. Estradiol also markedly increased the ability of the cells to grow both in suspension and semisolid Methocel cultures. In suspension, the cells grew as tight balls which clustered to gether to give small organoid-like structures reaching diame ters of 4 mm and composed of an outer shell of living cells containing a central cavity of necrotic cells. In the absence of estradiol in both monolayer and suspen sion, the cells went through a limited and constant number of divisions and then stopped, such that the final cell number was determined by the initial plating density. In the presence of estradiol, this block was removed such that in monolayer cultures the final cell number was independent of plating den sity. A major loss of estradiol response was found if the cells were grown for 7 to 14 days in the absence of estradiol. This loss of response appeared to be due to a loss of ability to grow rather than to selective cell death within the population.

Effect of estradiol on human breast cancer cells in culture.

Abstract: Conditions are described for growing and maintaining the estradiol sensitivity of the human breast cancer cell line ZR-75-1 both in monolayer and suspension cultures. Either newborn calf or fetal calf serum can be used in the culture medium, but an effect of estradiol on growth of the cells was only observed reproducibly if the serum was first treated with dextran-charcoal. Sulfatase treatment of the sera prior to dextran-charcoal treatment did not decrease cell growth in the absence of added estradiol, indicating that estrogen sulfates are unlikely to contribute to cell growth in dextran-charcoal-treated sera. In monolayer cultures, estradiol increased both the growth rate and final saturation density of the cells for each individual plating density tested in a dose-dependent manner with maximal stimulation occurring between 10(-10) and 10(-8) M estradiol. Estradiol also markedly increased the ability of the cells to grow both in suspension and semisolid Methocel cultures. In suspension, the cells grew as tight balls which clustered together to give small organoid-like structures reaching diameters of 4 mm and composed of an outer shell of living cells containing a central cavity of necrotic cells. In the absence of estradiol in both monolayer and suspension, the cells went through a limited and constant number of divisions and then stopped, such that the final cell number was determined by the initial plating density. In the presence of estradiol, this block was removed such that in monolayer cultures the final cell number was independent of plating density. A major loss of estradiol response was found if the cells were grown for 7 to 14 days in the absence of estradiol. This loss of response appeared to be due to a loss of ability to grow rather than to selective cell death within the population.

Generation of monoclonal antibodies reacting with normal and cancer cells of human breast.

Abstract: From the fusion of the murine myeloma P3- X63-Ag8-U1 with spleen cells from a mouse immunized with the human breast cancer cell line MCF-7, 88 hybridomas producing antibodies reacting with the immunizing cells were obtained. After a first screening on human leukocytes, red blood cells, and platelets and on human and fetal calf serum, three monoclonal antibodies, MBr1, MBr2, and MBr3, with specificity for the immunizing cells were isolated and further characterized. The three monoclonals were tested by isotopic antiglobulin assay and immunofluorescence on a panel of normal cells or cell membrane preparations, including milk epithelial and foam cells; on plasma and milk proteins; on cells or cell membrane preparations from fresh surgical specimens of breast, kidney, and ovarian carcinomas; and on various tumor cell lines. MBr1 and MBr2 had a superimposable reactivity and showed specificity for a structure which seems to characterize both normal and neoplastic mammary gland epithelial cells.

A Novel Dye Exclusion Method for Testing in Vitro Chemosensitivity of Human Tumors

Abstract: Dissociated cancer cells are exposed to antineoplastic drugs (5 × 10 4 viable cells/drug for 1 hr or continuously) and cultured for 4 to 6 days in liquid medium. Cells are then stained with Fast green dye, sedimented onto slides with a Cytospin centrifuge, and counterstained with a modified hematoxylin and eosin technique. Dead cells stain with Fast green, and living cells stain with hematoxylin and eosin. Cell kill is calculated as percentage of control based on the relative numbers of living tumor cells, living non-tumor cells, and dead cells. Drug sensitivity could be assayed in 125 of 162 specimens of human neoplasms obtained from malignant effusions (16 of 18), excisional biopsies (31 of 44), needle biopsies (34 of 47), endoscopic biopsies (18 of 23), peripheral blood samples (19 of 20), and bone marrow aspirates (five of seven). The assays were successful (median of ten drugs tested) in: 46 of 64 adenocarcinomas; four of 11 squamous cell carcinomas; five of seven lymphomas; six of seven melanomas; two of four sarcomas; 18 of 20 transitional-cell carcinomas; 14 of 15 small-cell carcinomas; seven of eight myelomas; 12 of 12 chronic lymphocytic leukemias; seven of nine acute leukemias, and four of five "undifferentiated" carcinomas. The assay results demonstrated a strong correlation between the in vitro chemosensitivity of different types of tumors and the known clinical response patterns of these tumors. This assay can be used to determine which specific cells are killed in a heterogeneous cell population. Further work is needed to determine if this assay may be useful in blind screening trials for antineoplastic agents or if it may be of clinical use in predicting response to agents which are not cycle specific.

Antiestrogenic Effect of R5020, a Synthetic Progestin in Human Breast Cancer Cells in Culture*

Abstract: To see whether progestins prevent estrogen action in breast cancer cells, we have studied in vitro the effect of R5020 on the cell growth and the synthesis of secreted proteins in T47D and R27 human breast cancer cells While R5020 had no effect on cell growth when tested alone, it significantly inhibited the growth of both cell lines in the presence of estradiol (1 nM) The effect was most clear-cut after 10–12 days of treatment and was dose dependent, a half-maximal inhibition occurred with 1 nM R5020 R27, a cloned MCF7 variant resistant to Tamoxifen, remained responsive to R5020, which prevented the effect of 17β-estradiol (E2) and inhibited cell growth in the presence of Tamoxifen This suggests that the two antiestrogens are acting through different mechanismsDihydrotestosterone and dexamethasone did not reproduce or inhibit the effect of R5020 on cell growth R5020 was ineffective in a rat tumor cell line containing androgen and glucocorticoid receptors but lacking progesterone receptors and estro

Correlation between circulating cancer cells and incidence of metastases.

Abstract: Quantitative aspects of the behaviour of B16 melanoma and Lewis lung carcinoma cells during the post-intravasation stages of metastasis were examined in relation to their spontaneous metastatic potential. Cancer cells were isolated from the blood of mice bearing i.m. tumours throughout tumour growth using a novel discontinuous gradient centrifugation technique. Four times more Lewis carcinoma cells than B16 melanoma cells were shed into the circulation, although the numbers of cells shed from either tumour were orders of magnitude more than the numbers of spontaneous pulmonary metastases which developed. Greater numbers of Lewis carcinoma cells were also shed as clumps and with leukocytes attached. However, although similar numbers of radiolabelled melanoma and Lewis carcinoma cells were arrested in the lungs after i.v. injection, fewer carcinoma cells were retained there and 12 times fewer Lewis carcinoma nodules developed in the lungs following injection of non-radiolabelled cells. It appears that the low lung colonization potential of the Lewis lung carcinoma is compensated for during spontaneous metastases by the numbers of cells shed from primary tumours as single cells and as clumps.

Plasminogen Activator Secretion of Human Tumors in Short-Term Organ Culture, Including a Comparison of Primary and Metastatic Colon Tumors

Abstract: The secretion of plasminogen activator by explants of 92 human malignant tumors was studied in short-term organ culture. Where possible, adjacent normal tissue of the presumed origin of the tumors was also studied. The study included adenocarcinomas of the lung, colon, prostate, breast, and stomach and different types of sarcomas. In addition to the measurement of secretion rates, all tissues were quantitatively extracted to determine the amount of cell-bound enzyme. Both culture fluids and extracts were analyzed with respect to the type of plasminogen activator they contained by immunoinhibition with goat immunoglobulin G formed against purified human urinary urokinase sodium dodecyl sulfate:gel electrophoresis followed by zymography. The study yielded the following conclusions: ( a ) the measurement of plasminogen activator secretion rates gives a much sharper differentiation between malignant and normal tissues than does the amount of extractable enzyme; ( b ) the enzymes secreted in short-term organ culture are, in the great majority of the cases studied, of the urokinase type, even when a large fraction of the activator contained in the tissue is of the vascular type; ( c ) the secretion rates of metastatic tumors of the colon are much lower than those of the primary ones; ( d ) immunoperoxidase staining of tissue sections reveals that urokinase is localized predominantly in the tumor cells. The low secretion rates of metastatic tumors, probably a reflection of this property in the original cell that gave rise to the metastatic focus, could be of advantage to circulating cancer cells. Such cells would not dissolve the microthrombus thought to be essential for the arrest of cancer cells in the capillaries of target organs.

Cell detachment and metastasis.

Abstract: Cancer cell detachment in three distinct and critical parts of the metastatic cascade is discussed. The detachment of cancer cells from their parent tumors is an initial early event in metastasis. The site of detachment with respect to proximity to blood vessels may determine the initial dissemination route. Many factors affect cell detachment; we specifically consider the effects of growth-rate, necrosis, enzyme activity, and stress on cell release in terms of metastasis-promoting mechanisms. Detachment is also discussed in relation to active cancer cell locomotion, where localized detachment from the substratum is a prerequisite for translatory movement. The importance of active cell movement in tissue invasion has only recently been assessed, and, in the case of at least some human malignant melanomas, a zone of actively moving cancer cells is believed to precede the growing body of the tumor. The secondary release of cancer cells from temporary arrest sites at the vascular endothelium consequent upon intravascular dissemination is also a major area of investigation. Circulating cancer cells arrest at vascular endothelium or are impacted in small vessels, however, most are released into the circulation and subsequently perish. The blood stream is a hostile environment, and it is probable that cancer cells are sufficiently damaged in translocation by hemodynamic trauma and humoral factors such that they easily detach or are 'sheared-off' the vascular endothelium by blood flow. Another possibility is that in some cases they are processed by 'first organ encounters' and perish before or shortly after arriving in a second organ. Animal studies have shown that, following intravenous injection, 60-100% of the injected dose of viable cancer cells are initially arrested in the lungs, but very few remain after 24 hr. As it is only those retained cells which produce tumors, the mechanisms involved in this secondary release, which occurs in all organs so far examined, are critical to any understanding of the metastatic cascade and metastatic inefficiency. The arrest of cancer cells at the vascular endothelium and their subsequent release have been associated with the presence of platelets, and the deposition of fibrin and manipulation of platelet-aggregating mechanisms and fibrinolysis are discussed in terms of their antimetastatic effects. The role of the reticuloendothelial system, natural killer cells, and polymorphs is discussed in relation to cancer cell clearance from blood vessels and also to inherent cancer cell properties which may act to inhibit their metastasis. Although detachment of cancer cells from a primary tumor may be regarded as metastasis promoting, secondary release of cancer cells may be associated with inhibition of metastasis.

Immunochemical Analysis of the Determinant Recognized by a Monoclonal Antibody (MBr1) Which Specifically Binds to Human Mammary Epithelial Cells

Abstract: A monoclonal antibody (MBr1) raised against a membrane preparation (CM) of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated (S. Menard, E. Tagliabue, S. Canevari, G. Fossati, and M. I. Colnaghi. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res., 43:1295-1300, 1983), was used to biochemically define and partially purify its target antigen. The antigenic activity recognized by MBr1 was unaffected by treatment of MCF-7 cells with trypsin, protease K, or Vibrio cholerae neuraminidase and by heating at 100 degrees but was abolished by treatment with methanol. Since this behavior suggested a glycolipid nature of the MBr1-defined antigen, total lipids were obtained by chloroform:methanol or tetrahydrofuran:phosphate buffer extractions from crude membrane preparations of MCF-7 cells and of breast cancer surgical specimens. Total absorption of MBr1 activity was found by breast cancer lipid extracts, whereas no absorbing capability was detected with a series of highly purified acid and neutral glycolipids or with normal and neuraminidase-treated red blood cells of human, ox, and sheep species. The same pattern of inhibition of MBr1-binding activity was obtained with total lipid extract and both phases after diethyl ether partition. However, when the three extracts were chromatographed on diethylaminoethyl-Sepharose, the antigenic activity was recovered only in the neutral glycolipid fractions. Periodate oxidation of MCF-7 crude membrane preparation abolished MBr1-binding activity, suggesting that the carbohydrate portion of the molecule may constitute the antigenic determinant.

Monoclonal antibodies (F36/22 and M7/105) to human breast carcinoma.

Abstract: Cloned hybridoma cell lines were obtained from fusions of murine myeloma cells with lymphocytes of mice immunized against human breast cancer cells. Hybridomas F36/22 and M7/105 produced antibodies whose binding to breast cancer cells could not be inhibited by prior absorptions with fibroblasts, lymphoblastoid cells, or erythrocytes. Results from cell surface binding assays using a panel of tumor cell lines indicated that antibodies F36/22 and M7/105 recognized determinants expressed maximally on breast cancer cells. Antibody F36/22 reacted with normal mammary epithelial membranes and milk fat globule membranes, whereas antibody M7/105 produced no detectable binding to these specimens. Antigens carrying these epitopes each showed reactivity with concanavalin A lectin. The determinant corresponding to antibody F36/22 was detectable in histological sections of a subset of breast tumors obtained at surgery.

Cancer treatment by intracellular hyperthermia

Abstract: A treatment of cancer by the application of chemical reactions intracellularly capable of the intracellular generation of heat so as to induce selective thermal death of cancer cells in living tissue. Metabolizable minute particles of a size less than one micron are intravenously injected into the patient and absorbed by the cancer cells. The oxygen level of the patient's blood is then increased. The rate of intracellular chemical reaction in the cancer cells due at least in part to the intracellular presence of these minute particles is thereby increased and intracellular heat generated. The oxygen level is increased until the intracellular temperature has increased at least 8.0 degrees Centigrade but not more than 9.5 degrees Centigrade thereby selectively killing the cancer cells without damaging the normal cells.

Effects of Amiloride on Tumor Growth and Intracellular Element Content of Tumor Cells in Vivo

Abstract: The effects of amiloride, a reported inhibitor of serum-stimulated sodium influx, were tested on tumor growth, tumor cell proliferation, and intracellular element content of cancer cells in vivo. We have shown previously that cancer cells have high intranuclear levels of sodium compared to those of their normal counterpart cells and have postulated that such a high level of sodium may be involved in the cancer state. We now report that amiloride, when given in a series of injections, inhibited both H6 hepatoma and DMA/J mammary adenocarcinoma growth in vivo in a dose-dependent fashion and that 3 injections of amiloride at a dose of 1.0 microgram/g body weight into mice bearing H6 hepatomas resulted in a significant decrease in the intranuclear content of sodium but not the content of magnesium, phosphorus, sulfur, chlorine, or potassium as measured by electron probe X-ray microanalysis in the H6 hepatoma cells. Amiloride at dosages as low as 1.0 microgram/g body weight per injection also inhibited tumor cell proliferation as measured by the tritated thymidine autoradiography labeling index. Amiloride caused no changes in the mean profile diameters of metaphase or interphase H6 hepatoma or DMA/J mammary adenocarcinoma cells, suggesting that the action of amiloride on tumor growth was not due to cell volume changes. These data show that amiloride both inhibited tumor growth and decreased the proliferation of the tumor cells in the H6 hepatomas which was correlated with a decreased intranuclear sodium content.

Collagen Degradation by Metastatic Variants of Lewis Lung Carcinoma: Cooperation between Tumor Cells and Macrophages

Abstract: Interactions between cancer cells and host macrophages might have important regulatory roles in controlling the expression of the metastatic phenotype, particularly by regulating the production of proteases necessary for tissue invasion. To investigate that possibility, mouse macrophages and Lewis lung carcinoma (LLC) cells from four clonal subpopulations with either low or high metastatic ability were cultured on [14C]collagen (type I)-coated plates. They did not degrade collagen when they were cultured independently on that substrate, but they were induced to do so when macrophages and cancer cells were cultured together. An increased production of neutral collagenase and other neutral protease activities was observed simultaneously. The degree of stimulation of collagen degradation varied according to the cancer cell subpopulation present in the cocultures. For a given LLC cell subpopulation, similar degrees of stimulation of collagen degradation were achieved with either bone marrow-derived or resident peritoneal macrophages, either syngeneic (from C57BL/6 mice) or allogeneic; lower stimulations were obtained with thioglycolate-elicited peritoneal macrophages. Macrophage-conditioned culture media could be substituted for living macrophages to stimulate collagen degradation or collagenase secretion by LLC cells, but LLC cell-conditioned media did not stimulate collagen degradation by macrophages. This suggests that, in the cocultures, collagen degradation is achieved mainly by the cancer cells, not by the macrophages, and that it is induced by a soluble factor, a monokine, produced by the macrophages. That factor might be identical to a recently identified rabbit monokine that stimulates fibroblasts or synovial cells to degrade collagen and proteoglycan and to activate plasminogen, because rabbit macrophage-conditioned media containing that monokine also stimulated collagen degradation by LLC cells.

Calcitonin Effects on Growth and on Selective Activation of Type II Isoenzyme of Cyclic Adenosine 3′:5′-Monophosphate-dependent Protein Kinase in T 47D Human Breast Cancer Cells

Abstract: The influence of calcitonin on cell growth was examined in the human breast cancer cell line, T 47D. These cells possess specific high-affinity receptors for calcitonin as well as a sensitive calcitonin-responsive adenylate cyclase. In the T 47D cells, low doses of salmon calcitonin initially stimulated cell growth and the incorporation of [3H]thymidine into acid-insoluble macromolecules. This initial stimulation was followed by an inhibitory effect of calcitonin upon cell proliferation, which occurred during the log phase of growth, was dose dependent, and resulted in prolongation of doubling time from 36 to 90 hr. DNA and protein content correlated well with cell number. By 7 to 9 days of treatment, cell numbers of calcitonin-treated cells reached a mean of 66.5 ± 3.7% of control ( p < 0.001, n = 8) (range, 51.3 to 82.9%). This biphasic effect of calcitonin on T 47D cells was reproduced by human calcitonin and prostaglandin E2 in the order of potency with which they influence adenylate cyclase. Epidermal growth factor (10−9 m) and insulin (10−9 m) stimulated the growth of T 47D cells, but this effect was abolished when either hormone was combined with salmon calcitonin (3 × 10−10 m). Calcitonin specifically activated type II isoenzyme of cyclic adenosine 3′:5′-monophosphate-dependent protein kinase in the T 47D cells. In view of other published data relating activation of this isoenzyme to growth regression in cancer cells, this response to calcitonin may be causally related to the inhibitory effect of the hormone upon cell replication in T 47D cells. The mechanism of the early stimulatory effect of calcitonin upon mitogenesis is not explained, although the possibility of stimulation of activity of type I isoenzyme of cAMP-dependent protein kinase has not been entirely excluded in the present experiments.

Immunohistochemistry of Gastric Carcinomas and Associated Diseases: Novel Distribution of Carcinoembryonic Antigen and Secretory Component on the Surface of Gastric Cancer Cells'

Abstract: Carcinoembryonic antigen (CEA) and secretory component (SC) were localized by peroxidase-labeled antibody immunocytochemistry in normal and abnormal human gastric mucosa. In normal epithelium, both glycoproteins were absent or only faintly present, but in intestinal metaplasia and carcinoma both were prominently present. CEA and SC on the surfaces of metaplastic epithelial cells were polarized. That is, CEA was expressed only on the microvillous surface and SC was expressed only on the basolateral surface. In gastric cancer, CEA and SC were distributed over the entire surface of the neoplastic cells. Thus, deviations from the normal differentiation and maturation of gastric epithelial cells were accompanied by abnormalities in surface expression of CEA and SC. These observations, together with compatible observations previously made in colonic neoplasia (DJ Ahnen, PK Nakane, and WR Brown, Cancer 49:2077, 1982), suggest that loss of polarity of surface membrane components is a characteristic of neoplastic epithelial cells.

Two new estrogen-supersensitive variants of the MCF-7 human breast cancer cell line.

Abstract: Two new estrogen-sensitive variants of MCF-7 human breast cancer cells, CG-4 and CG-5, are described in this report. These cells were derived from a casual contamination by MCF-7 cells of primary cultures from a human adenocarcinoma of the breast and a pleural effusion of a patient with advanced breast cancer, respectively. Careful characterization of these variants revealed chromosomal properties highly similar to and alloenzyme phenotypes identical to those of MCF-7 cells which were simultaneously cultured in the laboratory. MCF-7, CG-4 and CG-5 cells were tested for estrogen responsiveness under identical growth conditions, that is in the presence of culture medium supplemented with 5% charcoal-treated serum. While the number of MCF-7 cells increases by about 40% over the controls in the presence of physiological concentrations of estradiol, the number of CG-4 cells doubles and the number of CG-5 cells triples. All these cell lines have approximately the same number of estrogen, androgen, glucocorticoid, and progesterone receptor sites/cell. Since several difficulties arise in demonstrating the estrogen responsive growth stimulation of currently available human breast cancer cells, these two new variants, characterized by a high and reproducible estrogen responsiveness, afford a new model for studying the mechanisms by which estrogen regulates cell proliferation. The problems related to the careful characterization of every established cell line are discussed.

Liver-to-lung traffic of cancer cells.

Abstract: Following the injection of B16 melanoma cells into the portal veins of mice, all animals developed liver tumors, but only 16% developed lung tumors Portal vein injections of radiolabelled B16 and Walker 256 cancer cells into mice and rats, respectively, revealed that all of the cells were temporarily arrested in the liver and most were then slowly released Bioassays indicated that of 8 X 10(4) B16 cells released from the liver over 24 h after portal vein injections of 10(5) cells, only approximately 1% were delivered to the lungs in a viable state Experiments made with radiolabelled B16 cells showed that transit through either the liver or lungs following portal vein or tail vein injections, respectively, resulted in massive death of cancer cells It is suggested that the death of most circulating cancer cells passing through the first organ encountered after leaving their primary tumor, serves to severely limit their further direct spread to other organs It is therefore expected that metastases to these other organs would, to a large extent, be generated by cancer cells from metastases in the "first organs" as distinct from direct seeding from cancer cells released from the primary tumor If the results of the present experiments have general application, they serve to emphasize the importance of metastasis of metastases in the natural history of the spread of cancer In a previous publication (Weiss, 1980), studies of lung-to-liver traffic of cancer cells in rats revealed that, after tail vein injections, most Walker 256 cells were temporarily arrested in the pulmonary vasculature and then slowly released A large proportion of the released cancer cells were dead or lethally injured on release from the lungs, and this "first organ processing" apparently accounted for the comparative rarity of extrapulmonary tumors following tail vein injections In this communication, the concept of "first organ processing" of circulating cancer cells is further examined with respect to the liver-to-lung traffic of B16 melanoma and Walker 256 cells injected into the portal veins of mice or rats respectively Both of these cell types grow well in the lungs following tail-vein injection

Receptor status and subsequent sensitivity of subclones of MCF-7 human breast cancer cells surviving exposure to diethylstilbestrol.

Abstract: The estrogen receptor (ER)-positive human breast cancer cell line MCF-7 was incubated continuously in the presence of pharmacological concentrations of diethylstilbestrol (DES) in an attempt to correlate receptor status with DES sensitivity. It was consistently observed that cytotoxicity occurred at DES concentrations greater than 5 X 10(-6)M; however, a small percentage of cells, both from the wild-type MCF-7 line and from subclones derived in soft agar from single MCF-7 cells, survived, with altered morphology, up to 4 months of continuous exposure to DES concentrations ranging from 5 X 10(-6) to 1 X 10(-4)M. Characterization of seven regenerated surviving cell populations suggested that they remained ER positive; no evidence could be found for a block in the pathway of hormonal activation, as determined by progesterone receptor induction, to explain the ability of these cells to survive DES. Three regenerated cell populations were reexposed to DES. Two remained as sensitive to growth inhibition as untreated parent cells from which they were derived; however, one of these, designated MCF-7(35-1), was found to have autonomously high progesterone receptor (463 +/- 94 fmol/mg of cytosol; Kd = 1.8 +/- 0.2 X 10(-9)M) which was not significantly stimulated by the addition of 1 X 10(-8)M 17beta-estradiol for 72 hr. The third population, designated MCF-7(35-3), which survived initial exposure to 5 X 10(-5)M DES for 109 days and which remained ER positive (27 +/- 3 fmol/mg of cytosol; Kd = 0.8 +/- 0.2 X 10(-10)M) and progesterone receptor inducible, demonstrated significantly decreased sensitivity (p = 0.025) on reexposure to DES; conversely, significantly increased sensitivity (p less than 0.03) to the antiestrogen tamoxifen was observed. The mechanisms by which some MCF-7 cells survive prolonged exposure to DES are not certain; the data suggest that there is no clear relationship between ER status and sensitivity to DES and that there is no way of predicting the ultimate status of cells surviving DES treatment.

Growth factors, cell proliferation and cancer: an overview.

Abstract: In recent years it has been recognized that cell proliferation is controlled by a variety of mitogenic molecules some of which are becoming available in highly purified form. Studies carried out with combinations of defined growth-promoting molecules under chemically defined conditions have revealed an important aspect of their action: the existence of potent synergistic effects. By virtue of such synergistic effects, specific combinations of mitogenic hormones can be as effective as whole serum in initiating and supporting proliferation of many cell types. These developments are having a considerable impact on the culture of certain normal or tumour cells, on the design of assays for novel mitogenic molecules and on the interpretation and execution of experiments directed to elucidate the molecular events leading to cell proliferation. In particular, the availability of pure mitogenic molecules has opened up the possibility of exploring the molecular and physiological properties of the cellular receptors related to growth control, and the nature of the intracellular signals (e.g. ion fluxes, cyclic nucleotides and cytoskeletal changes) capable of eliciting or modulating a mitogenic response. Thus, growth regulation can be formulated in terms of external signals, cellular receptors and intracellular signals as depicted in Figure 1. Finally, growth factors appear to have a role in the development and expression of malignant transformation because tumour promoters and mitogenic hormones share common pathways in eliciting mitogenesis in certain cell types, and because tumour cells produce potent growth-promoting polypeptides. Production of growth factors by tumour cells raises fundamental questions concerning the role of these molecules in the direct causation of the unregulated growth of the cancer cells. It can only be a matter of time before the availability of highly purified tumour-derived growth factors will allow the determination of their structure, mechanism of action and the development of immunologic assays to monitor their presence in tumour-bearing animals or cancer patients.

Translocation of cytoplasmic estrogen receptors to the nucleus: immunohistochemical demonstration utilizing rabbit antibodies to estrogen receptors of mammary carcinomas.

Abstract: Rabbit antibodies to cytoplasmic estrogen receptors (ER) of human breast carcinoma were utilized for investigating steroid-triggered in-vitro translocation of cytoplasmic ER to the nuclear compartment of the estrogen target cells. The immunofluorescent method (IF) previously described (S Raam et al., Eur J Cancer Clin Oncol 18: 1–12, 1982) was employed for immunohistochemical localization of ER. Four cases of normal endometrium, two cancers of the endometrium, and MCF-7 human breast cancer cells were maintained in a steroid free medium and exposed at 37°C for two hours to growth medium alone (control) or to 2.5, 25 or 250 nanomoles of estradiol (E2), diethylstilbestrol (DES), or monohydroxytamoxifen (OH-TX). At the end of the incubation period the cells were processed for intracellular localization of ER. Complete traslocation of IF from the cytoplasm to the nuclear compartment was evident in all normal endometrial cells exposed to E2, DES or OH-TX for two hours. While cells from the endometrial cancer ‘S’, like the normal cells, translocated IF to the nucleus, cells of another cancer (‘KLE’) failed to translocate when exposed to E2 or OH-TX. Partial translocation was evident in ‘KLE’ cells exposed to DES. In MCF-7 cells grown in the absence of E2, IF was exclusively cytoplasmic. When these cells were exposed to the hormones, 50% showed a complete transfer of IF to the nucleus; in 40% a delayed response was evident; 10% failed to translocate. The results revealed the suitability of anti-ER antibodies for investigating the intracellular dynamics of ER in target cells responding to estrogens or antiestrogens.

Role of polymorphonuclear leukocytes in the pulmonary clearance of arrested cancer cells.

Abstract: Treatment of mice with trypan blue led to an accelerated clearance of 125I-5-iodo-2'-deoxyuridine-radiolabeled B16 melanoma cells arrested in the lungs of tumor-bearing mice and the numbers of pulmonary nodules developing after intravenous injection of nonradiolabeled melanoma cells was reduced. Although macrophage function was normal or depressed in trypan-blue-treated mice, there were increased numbers of circulating polymorphonuclear leukocytes which had increased cytotoxicity for B16 melanoma cells and which produced increased levels of toxic oxygen radical. These findings suggested that polymorphs were responsible for accelerated pulmonary clearance of arrested melanoma cells and this conclusion was supported by the decreased clearance of cancer cells from the lungs of mice treated with antipolymorphonuclear leukocyte antiserum. These studies expand the repertoire of effector cells responsible for determining the extent to which hematogenously disseminated cancer cells are retained in the pulmonary vasculature prior to their extravasation and secondary growth.

Doxorubicin (Adriamycin) transport in Ehrlich ascites tumour cells: comparison with transport in human red blood cells.

Abstract: The doxorubicin (Adriamycin) transport was investigated in murine Ehrlich ascites tumour cells by measuring the initial rate of cellular net uptake in vitro at 37°C (pH 7.3). Transport characteristics were compared with previously published data on doxorubicin transport in human red blood cells. The apparent permeability coefficient in ascites cells (2.4 × 10−5 cm sec−1) and in red cells was of the same order of magnitude when calculated from the initial influx into cells suspended in a salt solution (37°C, pH 7.3). Doxorubicin was strongly adsorbed to the cell surface of ascites cells in contrast to the doxorubicin adsorption to red cells when the cells were suspended in a salt solution. The adsorbed doxorubicin could be removed by washing the ascites cells either with DNA or with human albumin salt solutions indicating that the adsorption to cell surface components was reversible. Cell membrane modifiers, 1-alcohols, local anaesthetics, phloretin, HgCl2, para-chloromercuribenzoate, affected doxorubicin ...

Carcinoembryonic antigen production in human gastric cancer cell lines in vitro and in nude mice.

Abstract: A quantitative study of carcinoembryonic antigen (CEA) was made in nine human gastric cancer cell lines. Six of them were found to produce CEA in vitro. The production of CEA in the three cell lines derived from well differentiated tubular adenocarcinomas began at the mid-exponential phase of cell growth and reached its peak at the late stationary phase, the amount of CEA per 10(5) cells and the frequency of CEA-positive cells on immunostaining increased with culture time. In contrast, CEA in the three cell lines derived from poorly differentiated adenocarcinomas including a signet-ring cell carcinoma was produced immediately after plating and the amount of the antigen per 10(5) cells and the frequency of CEA-positive cells were almost constant throughout the cell growth phases. Serum CEA content in nude mice was low or not detectable in the case of subcutaneous heterotransplantation of gastric cancer cells, irrespective of CEA productivity of the cell lines in vitro. Intraperitoneal inoculation, however, led to high CEA levels in sera of nude mice bearing human gastric cancers. No significant difference was found between the two kinds of inoculation in terms of the total tumor weight and the frequency of CEA-positive cells in tumor tissues. One reason for the above findings may be that the transport of CEA in the subcutaneous tumors to the systemic blood flow is hindered.