Coordinate inhibition of plasminogen activator and tumor growth by hydrocortisone in mouse mammary carcinoma

A body of evidence has suggested that hormones which modulate plasminogen activator production by cultured tumor explants in vitro may have a qualitatively comparable effect on the growth of the same tumors in vivo. As a test of this correlation and to explore its potential for predicting the in vivo response of tumors to hormones, we have studied here the effect of hydrocortisone on the growth of primary and first generation transplants of mouse mammary tumor virus-determined mammary tumors in BALB/c X DBA/8 F1 (hereafter called CD8F1) mice; hydrocortisone had been found previously to inhibit plasminogen activator production by explants of these tumors. The results were: (a) hydrocortisone reversibly blocked the growth of palpable primary tumors; growth resumed at control rates following withdrawal of exogenous hormone; (b) hydrocortisone inhibited the growth of first-generation tumor transplants when administered either before or after the appearance of palpable tumors; (c) pretreatment with hydrocortisone both delayed the appearance of primary tumors and greatly reduced tumor incidence in susceptible mice; a substantial part of the decrease in tumor incidence was apparently irreversible; (d) hydrocortisone reduction of tumor growth was accompanied by inhibition of tumor plasminogen activator content, and these effects displayed a similar dose dependence (enzyme content of tumor lysates was measured by the 125I-fibrin plate assay); the enzyme present in control and hormone-treated tumors was predominantly of the urokinase type. These findings suggest that plasminogen activator production and mammary tumor growth in CD8F1 mice are coordinately regulated and thus encourage the view that plasminogen activator might be useful as an in vitro marker for predicting the in vivo response of tumors to hormones.     

Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied under a variety of conditions. In medium containing 10 microM thymidine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 microM thymidine and 50 microM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidylate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 microM thymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 microM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular tetrahydrofolate pool.     

Establishment and characterization of a mouse FM3A cell mutant resistant to topoisomerase II-inhibitor NC-190

We established an NC-190-resistant cell line, FM/NC-R, from the murine mammary carcinoma cell line FM3A and examined some of its characteristics. FM/NC-R cells were prepared by mutagen treatment followed by exposure to NC-190 in the culture medium. FM/NC-R cells were 76.5 times more resistant against NC-190 than FM3A cells as measured by their growth in vitro. FM/NC-R cells also showed cross-resistance to etoposide with NC-190. Neither NC-190 nor etoposide increased the lifespan of FM/NC-R-bearing mice at doses that prolonged the lifespan of FM3A-bearing mice more than four times. This resistance was not due to the change in the concentration of NC-190 in the cells, and there was no change in the expression of P-glycoprotein, a drug efflux pump in the cells. NC-190 and etoposide are inhibitors of DNA topoisomerase II, but there was no difference in cellular content of DNA topoisomerase II between the two cell lines as determined by Western blot analysis. The stabilization of DNA-DNA topoisomerase II cleavable complexes induced by NC-190 was lost in FM/NC-R cells. It was found that Gly881, which is located in the ATP binding site, was replaced by Arg in topoisomerase IIalpha of FM/NC-R cells. These results indicate that the NC-190-resistant cell line FM/NC-R contains a mutated DNA topoisomerase IIalpha.     

Establishment of mutant FM3A murine mammary carcinoma cell strains transformed with the herpes simplex virus type 1 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of anti-herpetic nucleoside analogs, mutants were constructed from murine FM3A mammary carcinoma cells, which were deficient in both thymidine kinase (EC 2.7.1.21) and thymidylate synthase (EC 2.1.1.45), but were transformed with a recombinant plasmid DNA containing the herpes simplex virus type 1 thymidine kinase gene. The transformed cells expressed viral thymidine kinase activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic agent (E)-5-(2-iodovinyl)-2'-deoxyuridine, which was only weakly inhibitory to the growth of the parent cells.     

Adenoviral transfer of xenogeneic MHC class I gene results in loss of tumorigenicity and inhibition of tumor growth

The immune system confers protection against a variety of pathogens and contributes to the destruction of neoplastic cells. Foreign major histocompatibility complex (MHC) protein serves as a potent stimulus to the immune system. In this report, a mouse H-2Kb gene was introduced into two poorly immunogenic tumor cell lines, a mouse colonic carcinoma cell line, MCA-26 (H-2Kd), and a rat mammalian carcinoma cell line, LN-4, in an effort to stimulate tumor rejection. Our results showed that the expression of xenogeneic MHC class I antigen completely abolished the LN-4 tumorigenicity in rats, whereas the expression of allogeneic MHC class I antigen only partially reduced the MCA-26 tumorigenicity in mice. Rats with tumor regression of LN-4/H-2Kb developed a T helper type 1-dominant response, whereas rats with LN-4 tumor growth developed a T helper type 2-dominant response. The immunized rats that experienced LN-4/H-2Kb tumor regression further developed protective immunity against a subsequent challenge of LN-4 cells. This protective immunity was mediated by the LN-4 tumor-specific cellular immune response against both the transduced and the parental LN-4 cells. Recombinant adenoviral vectors are highly efficient at in vitro and in vivo gene delivery. The LN4 cells transfected with the recombinant adenovirus AdV-H-2Kb in vitro expressed the cell surface H-2Kb molecule by fluorescence-activated cell sorter analysis. Adenovirus-mediated H-2Kb gene transfer in vivo can further significantly inhibit pre-established LN-4 tumors. Those rats with complete tumor regression further developed protective immunity against the subsequent challenge of a parental LN-4 tumor. Therefore, our study indicates that the adenovirus-mediated transfer of xenogeneic MHC class I gene may be an effective alternative to the current protocol of cancer gene therapy in which the allogeneic MHC class I gene is used.     

Dominant-negative activity of a Brca1 truncation mutant: effects on proliferation, tumorigenicity in vivo, and chemosensitivity in a mouse ovarian cancer cell line

In order to generate an in vitro mouse model for the study of human ovarian cancers, we compared the effects of a truncated Brca1 mutant expression on cellular phenotype with those of a full-length sense and antisense Brca1 expression in the ID-8 mouse epithelial ovarian cancer cell line. The examined cellular processes include proliferation, tumorigenicity in syngeneic mice in vivo and sensitivity/resistance to several cytotoxic drugs. We found that the expression of a spontaneous truncated Brca1 mutant in ID-8 cells which contain two endogenous wild-type Brca1 alleles led to a dominant-negative effect of Brca1, demonstrated by an increase in tumorigenicity in vivo and in chemosensitivity. Expression of a truncated Brca1 mutant in a mouse epithelial ovarian cancer cell line could thus provide a powerful in vitro model for the study of human BRCA1-related ovarian tumorigenesis.     

Inhibition of growth and linoleate-enhanced metastasis of a transplantable mouse mammary tumor by indomethacin

The influence of the cyclo-oxygenase inhibitor, indomethacin (IM), on the metastasis, development and prostaglandin E (PGE) levels of line 4526 mammary tumors grown in mice fed high fat (HF, 20%, w/w) diets containing various levels of linoleic acid (18:2) was investigated. Control mice that grew primary tumors and were fed HF diets containing 12% 18:2 (w/w) had 2-3 times the number of lung metastases than mice fed 1%, 4%, or 8% 18:2. Chronic treatment of mice with 10 micrograms/ml IM in drinking water reduced metastasis in 1% and 4% 18:2-fed mice compared to controls and completely inhibited the increased metastasis of mice fed the 12% 18:2 diet. Treatment with IM also increased the latency and decreased the growth rates of primary 4526 tumors of all dietary groups. Treatment of mice with a higher dosage of IM (20 micrograms/ml), decreased tumor metastasis even further compared to controls, but did not decrease tumor growth rate compared to the lower dosage of IM (10 micrograms/ml). Tumor PGE levels, measured by radioimmunoassay (RIA), were decreased by IM treatment. These data provide evidence that arachidonic acid metabolites such as PGE may be involved in the metastasis of 4526 mammary tumors.     

Possible mechanism of growth inhibition by Scutellaria baicalensis in an estrogen-responsive mouse tumor cell line

We have studied the effects of Saiboku-to, a traditional Chinese medicine having suppressive activities for leukotriene production and release, on the proliferation of the estrogen-responsive mouse Leydig tumor cell line B-1F. In our previous reports, it is shown that Saiboku-to promotes, but Scutellaria baicalensis, one of the components (herbs) of Saiboku-to, significantly inhibits the proliferation of B-1F cells in?vitro and in?vivo, and induces DNA fragmentation and morphological changes such as nuclear aggregation and fragmentation. In this study, we examined telomerase activity, cell cycle, polyunsaturated fatty acid metabolism and expression of nuclear factor κB (NF-κB) in order to determine the mechanism of growth inhibition in B-1F cells treated with Scutellaria baicalensis. Telomerase activity was decreased in a dose-dependent manner in treated B-1F cells. Cellular populations in the sub-G0/G1 and G2/M phases were increased, but those in M phase had no change. Although cyclin D1 mRNA was highly expressed in the presence of estradiol (E2), cyclin A and E mRNA levels did not significantly change. When B-1F cells were treated with Scutellaria baicalensis, expression of cyclin D1 was suppressed and that of p21 was inversely increased. Moreover, Scutellaria baicalensis influenced arachidonic and linoleic acid metabolism, and increased production of 13(S)-HODE. In the presence of E2 Scutellaria baicalensis decreased expression of NF-κB p65 to 0.71-fold in B-1F cells. These results show that Scutellaria baicalensis might induce cell cycle arrest at G1 phase and apoptosis via inhibition of telomerase activity, changes of enzymatic activities in polyunsaturated fatty acid metabolism and suppression of NF-κB.     

Induction of apoptosis in androgen-independent mouse mammary cell line by 1, 25-dihydroxyvitamin D3

Androgen-dependent tumors eventually progress to independent-tumors after androgen withdrawal. Effective treatment for hormone-independent tumors is therefore needed. Androgen-independent CS-2 cells could grow in serum-free culture whether androgen is present in the medium or not. In the present study, the mechanism of cell death in CS-2 cells was examined after 1, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃] treatment. 1, 25(OH)₂D₃ has been examined as an anti-tumor agent, but its role in promoting cell death is poorly understood. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, the cells underwent apoptosis with 1, 25(OH)₂D₃treatment. Northern-blot analysis was used to identify a series of genes whose expression per cell is enhanced during the apoptotic pathway. In the apoptotic process induced by 1, 25(OH)₂D₃, mRNA expression of testosterone-repressed prostatic message 2, transforming growth factor β1, glucose-regulated 78-kDa protein and calmodulin increased. Flow-cytometric analysis showed that 1, 25(OH)₂D₃ treatment resulted in a block in G₀/G₁ of the cell cycle. These results demonstrate that androgen-independent CS-2 cells retain the ability to undergo apoptosis by 1, 25(OH)₂D₃. This system appears to be a good model for investigating apoptosis of hormone-independent cancer. © 1996 Wiley-Liss, Inc.     

Expression of a mouse mammary tumor virus cross-reacting antigen on the cell surface of T-47D,a human mammary cell line

An antigen immunologically cross-reacting with the murine mammary virus major glycoprotein (gp52) was found on the cell surfaces of a human breast cancer cell line (T-47D) and its subcloned derivatives. The presence of this antigen was established by various immunological methods: indirect immunoperoxidase technique on live and fixed cells, indirect radioimmunoassay of live cells and immunoscanning electron microscopy. We therefore propose the T-47D cell line and its subclones as an in vitro model system for studying the modulation of expression of this tumor-associated antigen.     

Protection against cyclophosphamide-induced alopecia and inhibition of mammary tumor growth by topical 1,25-dihydroxyvitamin D3 in mice

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)₂D₃ was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)₂D₃, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)₂D₃-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)₂D₃ alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)₂D₃ pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)₂D₃ can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)₂D₃ was shown, either alone or in combination with CTX, to inhibit tumor growth. Int. J. Cancer 75:303–309, 1998. © 1998 Wiley-Liss, Inc.     

Inhibited growth in vivo of a mouse pregnancy-dependent mammary tumor (TPDMT-4) by an antiestrogen, 2alpha, 3alpha-epithio-5alpha-androstan-17beta-ol (10275-S).

TPDMT-4 mammary tumors, characterized by growth during pregnancy and postpartum regression, grew continuously in DDD virgin mice carrying pituitary isografts or 17 beta-estradiol plus progesterone pellets. In the experimental models, effects of 2alpha,3alpha-epithio-5alpha-androstan-17beta-ol(10275-S), testosterone, and ovariectomy were investigated. When tumors implanted with pituitary isografts into the fat pad reached palpable volumes, animals were ovariectomized or received 5 s.c. injections of 100 mug to 1 mg 10275-S or 300 mug testosterone weekly. Tumors regressed immediately after ovariectomy or after the start of 10275-S treatment and after approximately the 10th day of testosterone treatment. Cystadenomatous morphology with strong secretory activity was noticed in treated animals. Tumor growth induced by 17beta-estradiol plus progesterone pellets was inhibited completely or partially by 3 s.c. injections of 50 to 500 mug 10275-S or 100 mug testosterone weekly starting from the day after tumor and 17beta-estradiol plus progesterone pellet implantation.     

Control of mammary tumor cell growth in vitro by novel cell differentiation and apoptosis agents

The use of breast tumor differentiating agents to complement existing therapies has the potential to improve breast cancer treatment. Previously we showed quinidine caused MCF-7 cells to synchronously arrest in G1 phase of the cell cycle, transition into G0 and undergo progressive differentiation. After 72–96 h cells became visibly apoptotic. Using several analogs of quinidine we determined that MCF-7 cell cycle exit and differentiation are typical of quinoline antimalarial drugs bearing a tertiary amine side chain (chloroquine, quinine, quinidine). Differentiated cells accumulated lipid droplets and mammary fat globule membrane protein. Apoptosis was assayed by a nucleosome release ELISA. Quinidine and chloroquine triggered apoptosis, but not quinine, a quinidine stereoisomer that displayed weak DNA binding. The apoptotic response to quinidine and chloroquine was p53-dependent. A 4–15-fold induction of p21(WAF1) protein was observed in cells treated with quinidine or chloroquine prior to apoptosis, but p21(WAF1) was not increased in cells that differentiated in response to quinine. Chloroquine was most active in stimulating MCF-7 apoptosis, and quinine was most active in promoting MCF-7 cell differentiation. We conclude, distinct mechanisms are responsible for breast tumor cell differentiation and activation of apoptosis by quinoline antimalarials. Alkylamino-substituted quinoline ring compounds represented by quinidine, quinine, and chloroquine will be useful model compounds in the search for more active breast tumor differentiating agents.     

The role of metabolism in mammary epithelial cell growth inhibition by the isoflavones genistein and biochanin A

The basis for the differential sensitivity of cultured normalhuman mammary epithelial (HME) cells and a transformed humanbreast cancer MCF-7 cell line to growth inhibition by the isoflavonegenistein and its 4'-methyl ether derivative, biochanin A, wasexamined. In HME cells genistein is 5-fold more potent as agrowth inhibitor than biochanin A, whereas in MCF-7 cells biochaninA and genistein are equally potent as growth inhibitors. Basedon its properties as an in vitro protein tyrosine kinase (PTK)inhibitor, biochanin A would be expected to be a less potentgrowth inhibitor than genistein. To determine whether isoflavonemetabolism could account for the observed differences in growthinhibition, metabolism experiments were conducted with HME andMCF-7 cells using [4-¹⁴C]genistein and [4-¹⁴C]biochanin A. MCF-7cells extensively metabolized both isoflavones, producing twogenistein metabolites with molecular weights of 350 and 380and three biochanin A metabolites with molecular weights of270, 350 and 380. In contrast, significant genistein or biochaninA metabolism was not observed in HME cells. Using mass spectrometryand nuclear magnetic resonance analysis, metabolite 350 fromgenistein and biochanin A experiments was identified as genistein7-sulfate; biochanin A metabolite 270 was identified as genistein.Metabolite 380 was not unequivocally identified, but appearedto be a hydroxylated and methylated form of genistein sulfate.In MCF-7 cells, genistein 7-sulfate and metabolite 380 weredetected primarily in the cell media fraction, suggesting thatonce formed these polar metabolites were excreted from the cells.These data show that isoflavone metabolism by transformed breastepithelial cells modulates the growth inhibitory effects ofgenistein and biochanin A. In MCF-7 cells, genistein metabolismwas correlated with a decrease in growth inhibition, whereasbiochanin A metabolism was associated with an increase in growthinhibition.