Involvement of breast epithelial-stromal interactions in the regulation of protein tyrosine phosphatase-γ (PTPγ) mRNA expression by estrogenically active agents

Background. Protein tyrosine phosphatase (PTP) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicate that estradiol-17 (E₂)-induced suppression of PTP may play a role in mammary tumorigenesis. Zeranol (Z), a nonsteroidal growth promoter with estrogenic activity that is used by the US meat industry, induces estrogenic responses in primary cultured breast cells and breast cancer cell lines. Methods. PTP mRNA expression in human breast tissues and cells isolated from surgical specimens of mammoplasty and breast cancer patients were detected and quantified by RT-PCR. Immunohistochemical staining was used to localize PTP in human breast tissues. Breast epithelial and stromal cells were isolated and co-cultured to determine the involvement of cell–cell interaction in the regulation of PTP mRNA expression by E₂ and Z. Results. PTP mRNA expression was lower in cancerous than in normal breast tissues. Both E₂ and Z suppressed PTP mRNA levels in cultured normal breast tissues by 80%, but had a lesser effect in cultured epithelial cells isolated from normal breast tissues. In the co-culture system, both E₂ and Z suppressed PTP mRNA to a greater degree in epithelial cells than in stromal cells. In whole breast tissues, PTP was immunolocalized to the epithelium. Treatment with E₂ or Z diminished PTP staining indicating reductions in PTP at the protein level. Conclusions. The results indicate that both E₂ and Z regulate PTP expression in human breast and that epithelial–stromal cells interaction is important in the regulation of PTP expression by estrogenically active agents.     

Brain surface invasion and metastasis of murine malignant melanoma variants

Mouse B16 melanoma sublines were selected sequentially for their abilities to colonize brain meninges and leptomeninges of C5713L/6 mice. After 14 selections subline 1316-B14b was established that formed significantly more brain tumor colonies than the parental B16 line. Examination of brains at various times after intravenous or intra-arterial injection of B16 cells by electron microscopy revealed that B14b melanoma cells lodged in small brain blood vessels, proliferated and invaded through vessel walls into brain parenchyma and also along small blood vessels at perivascular sites. Invasion into brain parenchyma was characterized by extension of melanoma cell filopodia resulting in fragmentation and sometimes enfulgment of glial and neural cells.Analysis of cell surface proteins of B16 melanoma sublines revealed increased exposure of a M_r 90 000 glycoprotein on the high brain-colonizing cells. Antibodies against the M_r 90 000 glycoprotein reacted with a variety of human melanoma cell lines and with some fetal and adult tissues, indicating that this melanoma-associated component is not species-, tumor- or tissue-specific. The glycoprotein could be a cell surface receptor important in the survival and growth properties of melanoma cells in brain microenvironments.     

Effect of Dose and Infusion Time on the Delivery of p-boronophenylalanine for Neutron Capture Therapy

Clinical trials of boron neutron capture therapy (BNCT) for glioblastoma multiforme are currently in progress using p-boronophenylalanine (BPA) as the 10B delivery agent. Enhancement of tumor boron uptake and/or the tumor-to-blood (T:B) boron concentration ratio would have the potential of significantly improving the therapeutic gain of BNCT. The effects of total dose, infusion time, and route of administration of BPA on tumor and blood boron concentrations were studied in rats bearing the 9L gliosarcoma. Increasing the total dose of BPA from 250 to 1000mg/kg, administered intravenously over a 2-h infusion period, resulted in an increase in tumor boron concentration from 30 to 70µg 10B/g, with a constant T:B boron concentration ratio of about 3.7:1. Similarly, extension of the infusion time from 2 to 6h, at a constant dose-rate of 125mg BPA/kg/h, resulted in an increase in tumor boron concentration from 30 to 80µg 10B/g, while, again, maintaining a constant T:B ratio of about 3.7:1. In contrast, intracarotid infusion of BPA for 1h at a dose rate of 125mg BPA/kg resulted in an increase in the tumor boron concentration from 26 to 38µg 10B/g with a corresponding increase in the T:B ratio from 3.5:1 to 5.0:1. The effects of these results on the therapeutic gain potentially achievable with BNCT are discussed.     

Alterations in cellular retinol metabolism contribute to differential retinoid responsiveness in normal human mammary epithelial cells versus breast cancer cells

The present study was undertaken to compare ROH growth responsiveness between normal human mammary epithelial cells (HMECs), estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cells, and assess whether this responsiveness is correlated with differences in ROH metabolism, particularly RA synthesis. HMECs were markedly more growth sensitive to a physiological dose of ROH than breast cancer cells, exhibiting a significant decrease in cell number by 48h and >70% decrease by 144h. In comparison, numbers of MCF-7s were only decreased 32% by 144h. MDA-MB-231 cells were not affected. However, HMECs and MCF-7 cells displayed similar growth responsiveness to 1M RA, while MDA-MB-231 cells were minimally affected. Although the initial rates and extent of ROH uptake were comparable among cell types, ROH levels in HMECs progressively decreased to 20% of the peak by 24h and 10% by 72h. In contrast, ROH levels in the cancer cells remained relatively constant through 48h. The decrease in HMEC ROH was attributable to greater metabolism as evidenced by rapid and predominant retinyl ester formation. HMECs also produced 5 times more RA from ROH than MCF-7s and 10 times more than MDA MB-231 cells. Our results demonstrate that normal HMECs are markedly more responsive to the growth inhibitory effects of ROH than breast cancer cells, and that this responsiveness is associated with greater ROH metabolism including greater RA synthesis. These data suggest that altered ROH metabolism may be a factor in breast cancer progression.     

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil

Summary Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 M LCV concurrently with or after 10 M FUra following MTX (1 M) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV schedulling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-³H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 M LCV with 10 M FUra after 1 M MTX resulted in significantly more ternary complex formation than did 1 M MTX before 10 M FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-³H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.Abbreviations MTX methotrexate - FUra 5-fluorouracil - LCV d, 1-N ⁵-formyltetrahydrofolic acid, calcium salt (folinic acid, leucovorin) - F dUMP 5-fluoro-2-deoxyuridylate - FUTP 5-fluorouidine-5-triphosphate - PRPP 5-phosphoribosyl-1-pyrophosphate - CH₂FAH₄ N ^5,10-methylenetetrahydrofolate - dUMP 2-deoxyuridylate - dTMP thymidylate - TS thymidylate synthetase - PBS phosphate-buffered saline - NaCl 8.0 g - KCl 0.2 g - Na₂HPO₄ 1.15 g - KH₂PO₄ 0.2 g in 1 1 H₂O, pH 7.4 - FdUrd 5-fluoro-2-deoxyuridine This work was supported in part by grant CH-145 from the American Cancer Society and by grants CA-27130 and CA-08341 from the National Cancer Institute. LLD was supported by Postdoctoral Training Grant S-T32-CA09085-08 from the National Institutes of Health and EC was the recipient of a Cancer Research Award from the American Cancer Society     

Levels of water-soluble antioxidants in astrocytoma and in adjacent tumor-free tissue

Summary The aim of the present study was to investigate the oxidative status in astrocytoma. Samples of brain tissue from the centre to the periphery of the tumor were obtained from 11 astrocytoma patients undergoing computer tomography-guided stereotaxic operation, who had been previously treated with the corticosteroid dexamethasone. Part of the sample was investigated histologically for clarification of tumor type, and the presence of neoplastic and non-neoplastic tissue and necrosis. The rest was used for the quantification of the antioxidants ascorbic acid, uric acid, glutathione and cysteine by high performance liquid chromatography, and for quantification of DNA. Levels of antioxidants were calculated as g/g fresh tissue and mol/g DNA, a parameter related to cell content. There was significantly more DNA in neoplastic samples than in nonneoplastic ones, indicating increased cell density. Uric acid (g/g fresh tissue) was significantly increased in neoplastic compared with non-neoplastic tissue, and levels were even higher in necrotic tissue. There were no significant differences between neoplastic and non-neoplastic tissue levels of ascorbic acid, glutathione or cysteine, expressed as g/g fresh tissue. However, when levels of these three compounds were expressed as mol/g DNA, i.e. taking into account the higher cell density, ascorbic acid, glutathione and cysteine were significantly reduced in neoplastic samples compared with non-neoplastic ones. Results thus show that there are differences between the antioxidant levels in astrocytoma and non-neoplastic tissue, providing additional support for the hypothesis that free radicals play a role in tumor growth.     

Irofulven Induces Apoptosis in Breast Cancer Cells Regardless of Caspase-3 Status*

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48h incubation at 1M irofulven ( 3×GI₅₀), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24h but decreased markedly after 48h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48h. Normal HMEC cells were refractory to 1M drug with only 3–9% fragmented DNA after 12–48h, although apoptosis was observed at drug levels >3M. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20M with nearly complete abrogation of apoptosis at 100M. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).     

Development of alkylating agent-resistant human tumor cell lines

Summary Survival curves and dose escalation studies of four representative human tumor cell lines exposed to the various alkylating agents are presented. With HN2, at a level of one log of cell kill there was a fivefold range in drug concentration required to achieve this degree of cell kill among the cell lines, from 4.5 M for the SL6 lung adenocarcinoma to 22 M for the SW2 small-cell lung carcinoma. Four logs of SCC-25 squamous carcinoma cells were killed by 100 M CDDP; however, there was only about one log of SL6 cells killed by 500 M CDDP. To kill one log of G3361 melanoma cells required 380 M 4-HC and to kil one log of SCC-25 cells required 24 M, approximately a 16-fold difference. The curves for cell kill by L-PAM appeared to be biphasic, with a break at about 100 M. There was about a threefold range in drug concentration required to achieve one log of cell kill with L-PAM, from 60 M in the SCC-25 cell line to 18 M in the SW2 line. To kill one log of SCC-25 cells required 295 M BCNU and to kill one log of SW2 cell required 120 M, about a 2.5-fold difference. The range of maximally tolerated HN2 concentrations were from 1200M for the SL6 cell line, 48 times the initial concentration, to 300 M for the SCC-25 line, 16 times the initial concentration. The G3361 line tolerated the highest level of CDDP, 1900 M, 48 times the initial concentration. The SCC-25 line, on the other hand, tolerated only 600 M, 30 times the initial concentration. The SL6 cell line maximally tolerated 36 times the initial concentration of 4-HC (1450 M), whereas the SCC-25 cell line tolerated only 18 times the initial concentration (720 M). The G3361 melanoma tolerated 1555 M, 30 times the initial concentration of L-PAM, and the SCC-25 cell line tolerated 700 M, 14 times the initial concentration. The SL6 cell line tolerated the highest concentration of BCNU, 4200 M, 24 times the initial concentration. The SCC-25 cell line tolerated 1450 M, 8 times the initial concentration. In all cases, the SCC-25 cell line was least able to tolerate exposure to increasing concentrations of alkylating agents. The SL6 and G3361 cell lines showed the greatest tolerance for increasing concentrations of alkylating agents. With maximal selection pressure, in terms of intensity and duration, 5-to 15-fold resistance at best could be produced to these alkylating agents. This contrasts with other drugs, indicates that alkylating agents are more like X-rays, and has implications for high-dose clinical treatments. The importance of these findings to the clinical treatment of cancer is discussed.Abbreviations NH2 Nitrogen mustard (mustragen) - CDDP cis-diamminedichloroplatinum (II) (cisplatin) - BCNU N,N-bis(2-chloroethyl)-N-nitrosourea - L-PAM L-phenylalanine mustard (melphalan) - 4-HC 4-hydroperoxycyclophosphamide - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - PBS phosphate-buffered saline This work was supported by NCI grant #5PO1-CA38493 and a grant from the Mathers Foundation     

Alcohol consumption and risk of breast cancer: a cohort study

Objectives: To study the association between alcohol consumption and breast cancer risk. Methods: A case–cohort analysis was undertaken within the cohort of 56,837 women who were enrolled in the Canadian National Breast Screening Study (NBSS) and who completed a self-administered dietary questionnaire. (The NBSS is a randomized controlled trial of screening for breast cancer in women aged 40–59 at recruitment.) The cohort was recruited between 1980 and 1985, and during follow-up to the end of 1993 a total of 1469 women in the dietary cohort were diagnosed with biopsy-confirmed incident breast cancer. For comparative purposes a subcohort consisting of a random sample of 5681 women was selected from the full dietary cohort. After exclusions for various reasons the analyses were based on 1336 cases and 5238 noncases. Results: When compared to nondrinkers the adjusted incidence rate ratios (95% confidence intervals) for those consuming>0 and 10g of alcohol/day, >10 and 20g/day, >20 and thinsp;30g/day, >30 and 40g/day, >40 and 50g/day, and >50g/day were 1.01 (0.84–1.22), 1.16 (0.91–1.47), 1.27 (0.91–1.78), 0.77 (0.51–1.16), 1.00 (0.57–1.75), and 1.70 (0.97–2.98), respectively; the associated p value for the test for trend was 0.351. Similar findings were obtained when analyses were conducted separately in the screened and control arms of the NBSS, in premenopausal and postmenopausal women, for screen-detected and interval-detected breast cancer, and by levels of other breast cancer risk factors. Conclusions: The results of this study suggest that alcohol consumption might be associated with increased risk of breast cancer at relatively high levels of intake.     

The combination of X-ray-mediated radiosurgery and gene-mediated immunoprophylaxis for advanced intracerebral gliosarcomas in rats

Rats with advanced, imminently lethal, 4mm diameter, left-sided intracerebral 9L gliosarcoma (9LGS), a well characterized malignant tumor with some similarities to human high-grade astrocytomas, were used as a therapy model 14 days post-implantation of 10⁴ cells. Such tumor-bearing rats die within two weeks (median, 6 days) thereafter if untreated. However, if these tumors are exposed on day 14 to 12–25Gy of an electron-equilibrated 6MV photon beam (radiosurgery), survival is extended about 5–6 fold to a median of 34 days, but long-term survival (> 1 year) is increased only to 18%. Multiple subcutaneous inoculations of radiation-disabled 9LGS cells post-radiosurgery (immunoprophylaxis) extended lifespan and long-term (> 1 year) survival minimally (median, 37 days; 25%, respectively). In sharp contrast, radiosurgery followed by multiple subcutaneous inoculations of radiation-disabled 9LGS cells that had been transfected with granulocyte macrophage colony stimulating factor (GMCSF), a cytokine with demonstrated immune-enhancing properties (i.e. gene-mediated immunoprophylaxis, GMIMPR) increased long-term survival to 67%. To our knowledge, these results are the first to show that the combination of photon radiosurgery and GMIMPR is effective for an advanced, imminently lethal brain tumor in a mammal. These data raise the possibility that GMIMPR following radiation therapy might prove effective for the treatment of some human malignant gliomas.     

Cellular pharmacology and antitumor activity of N-(p-azidobenzoyl)-daunorubicin,a photoactive anthracycline analogue

Summary We have previously utilized N-(p-azidobenzoyl)daunorubicin (NABD), a photoactive analogue of daunorubicin (DNR), to identify unique anthracycline-binding polypeptides in rodent tissues and in tumor cells. Using cultured P388 tumor cells, we have now compared the cellular pharmacology and antitumor activity of NABD with that of DNR. Although rapidly accumulated by cells, the intracellular concentration of NABD was less than 20% that of DNR at steady-state levels. The cellular uptake of both drugs by P388 cells was dependent on extracellular drug concentration in the medium and on temperature. The rapid efflux of NABD and DNR from P388 cells in drug-free medium was reduced at lowered temperature (0 °C). Cytofluorescence microscopy demonstrated that NABD was predominantly localized in the cytoplasm, in contrast to the nuclear localization of DNR. NABD produced dose-dependent inhibition of [³H]thymidine (IC₅₀=10.0 M) and [³H]uridine (IC₅₀=1.60 M) incorporation in P388 cells to a lesser degree than DNR ([³H]thymidine, IC₅₀=0.15 M and [³H]uridine, IC₅₀=0.70 M). Continuous exposure to NABD inhibited P388 cell proliferation with an IC₅₀ of 0.27 M, compared with an IC₅₀ of 0.017 M for DNR. NABD is a pharmacologically active, photoactive analogue of DNR, which possesses properties different from those of the parent drug but similar to those of other anthracycline analogues. Photoaffinity labeling studies with NABD may identify important cytoplasmic constitutents which interact with this type of anthracycline and perhaps with the anthracycline antibiotics in general.Abbreviations used NABD N-(p-azidobenzoyl)daunorubicin - DNR daunorubicin - D₂ daunorubicinol - NABD₂ N-(p-azidobenzoyl)daunorubicinol - dDa 7-deoxydaunorubicin aglycone - dD_2a 7-deoxydaunorubicinol aglycone - TLC thin-layer chromatography - HPLC high-pressure liquid chromatography - THF tetrahydrofuran - PBS phosphate-buffered saline - DMSO dimethylsulfoxide     

Tumor necrosis factor-alpha induces interleukin-6 production via extracellular-regulated kinase 1 activation in breast cancer cells

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are frequently produced by breast cancer cells. These interleukins promote osteoclast formation and may mediate osteolysis at the site of breast cancer bone metastases. Transforming growth factor- (TGF-), tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) up-regulate IL-6 and IL-11 production in a cytokine-dependent fashion in breast cancer cells, but very little is known about their intracellular signaling pathways in breast cancer cells. To study TGF-, TNF- and IL-1 regulation of IL-6 and IL-11 production in human MDA-MB-231 breast cancer cells, we established single cell clones stably expressing dominant negative (DN) forms of the mitogen-activated protein kinases p38 (p38/AF) or ERK1 (ERK1K71R). We show here, that while basal, TGF- and IL-1 induced IL-6 production was similar in parental cells and in pcDNA3 control, ERK1K71R and p38/AF clones, TNF- induced IL-6 production was blunted in the ERK1K71R clones. TGF- and IL-1, but not TNF-, induced IL-11 production in parental MDA-MB-231 cells. Similar findings were detected in clones stably expressing p38/AF and ERK1K71R, which did not change basal IL-11 production either. In conclusion, TNF- induced IL-6 production is mediated via ERK1 activation in MDA-MB-231 cells. These observations may be helpful in designing new anti-osteolytic therapies.     

Balance of cell proliferation and apoptosis in breast carcinogenesis

We determined the mitotic and apoptotic index through the spectrum of preinvasive ductal breast lesions to invasive carcinoma in search of disturbances in the proliferation/cell death balance in breast carcinogenesis. Seventytwo pure preinvasive ductal breast lesions (without invasive carcinoma) and 103 invasive breast carcinomas were used. The numbers of mitotic and apoptotic cells were microscopically counted in hematoxylin and eosin stained sections (MI and Al, respectively), and the ratio of the values of MI and AI was calculated for each individual case (M/A index).A distinction was made between well differentiated and poorly differentiated breast lesions, based on histological type and nuclear grade, to arrive at two plausible progression models for breast carcinogenesis. For the well differentiated breast lesions, the MI was rather equal for hyperplasias and well differentiated DCIS, but increased 6fold from DCIS to well differentiated invasive carcinoma. The AI remained in the same range, resulting in a 4fold increase of the M/A index. For the poorly differentiated breast lesions, a significant increase in MI and AI was found from hyperplasia to poorly differentiated DCIS. From DCIS to poorly differentiated invasive carcinoma, the MI increased significantly and the AI decreased 2fold (n.s.), resulting in a 2.5fold significant increase of the M/A index.In conclusion, the net increase of the number of cells in the transition from well differentiated preinvasive to well differentiated invasive carcinoma is accompanied by an increase of cell proliferation rather than decrease in apoptosis, suggesting that in these lesions, proliferation related mechanisms are most important in carcinogenesis and progression. In contrast, in poorly differentiated breast lesions, decreased apoptosis seems to be also important in carcinogenesis and progression. At present, we are gathering patients with invasive breast cancer who had a previous biopsy with a preinvasive lesion to obtain further more direct evidence for this hypothesis.     

Intracellular concentrations of anti cancer drugs in leukemic cells in vitro vs in vivo

Summary A comparison of intracellular concentrations of daunorubicin, doxorubicin and ara-C in myeloid blast cells was carried out in vivo and in vitro. In vivo, blood samples were obtained from 27 patients with acute nonlymphoblastic leukemia during and up to 4 days after drug infusion. Leukemic cells were isolated and drug concentrations were determined by HPLC. Before treatment, leukemic cells from 21 patients were isolated from blood and bone marrow, and in vitro incubations were done with anthracyclines for 1–3 h at concentrations of 0.1–1.0 M and with ara-C for 1 h to 5 days at concentrations of 0.5–5.0 M. The cells were cultured for 5 days, during which cell samples were taken for drug determination. The results showed that incubation with 0.2 M daunorubicin for 1 h and 0.2 M doxorubicin for 3 h and continuous exposure to 0.5 M ara-C gave intracellular concentration curves similar to those obtained in vivo. After 5 days' culture, the cytotoxic effect was determined by vital dye staining with fast green, the addition of an internal standard of fixed goose erythrocytes, cytospin centrifugation and counter-staining of living cells with haematoxylin/eosin (DiSC). Incubations at the above-mentioned concentrations exerted a cytotoxic effect of approximately 50%. We conclude that in mimicking the in vivo situation, it is important to consider differences in intracellular pharmacokinetics.     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

Self-Reported Dietary Habits, Overall Dietary Quality and Symptomatology of Breast Cancer Survivors: a Cross-Sectional Examination

Little information is available about the relationship between quality of life of women who have survived breast cancer (specifically, symptoms including those of menopause and depression) and the quality of their diet. In this cross-sectional study, 117 women with known primary breast cancer completed a self-administered food frequency questionnaire (FFQ) reflecting usual diet during the past year, a Survey of Feelings and Attitudes using the Center for Epidemiologic Studies Depression scale (CES-D) and a survey that includes menopausal symptoms among others common to women with a history of breast cancer. When women's responses to the FFQ were scored using the Healthy Eating Index (HEI), most often diets were evaluated as those that need improvement with a mean total HEI score of 67.2. With regard to the CES-D scores, study women averaged 9.5, with 19 women being classified as clinically depressed. HEI and CES-D scores were inversely related (=–0.22, p=0.02). A negative correlation was also observed between energy-adjusted calcium intakes and CES-D scores (=–0.19, p=0.04). Clinical depressed women had not only lower HEI scores and calcium intakes, but also lower grain and variety scores. Comparisons to national data for disease-free women and that available for those with breast cancer suggest that our study women consumed diets low in energy and dietary variety. Diet quality may be an important factor influencing the manifestation of depressive symptoms in breast cancer survivors or conversely, poorer diet quality may be an outcome of depression.     

Constitutive activation of pp125fak in newly isolated human breast cancer cell lines

Our laboratory has developed twelve human breast cancer cell lines from primary and metastatic sites. In this report we demonstrate that eight of eight breast cancer cell lines examined exhibit constitutively tyrosine phosphorylated and enzymatically active endogenous pp125fak when grown in monolayer. The activation status of pp125fak in breast cancer cells in monolayer is significantly elevated over that exhibited by normal mammary epithelial cells cultured under the same conditions. Constitutive activation of pp125fak is the only characteristic so far studied that all of these breast cancer cell lines have in common. In contrast to HBC cells, tyrosine phosphorylation of pp125fak in HME cells was low or absent in monolayer culture but was induced to high levels by culturing the cells in Matrigel. Thus tyrosine phosphorylation and activation of pp125fak is a regulated process in normal mammary epithelial cells, but is constitutive in breast cancer cells. Finally, analysis of the ability of normal human mammary epithelial cells and breast cancer cell lines to grow under anchorageindependent conditions indicated that normal human mammary epithelial cells rapidly and uniformly lost viability when not substrateattached, whereas all of the breast cancer cell lines survived for a 3week culture period. Furthermore, a subset of the breast cancer cell lines grew to form large colonies under anchorageindependent conditions. Interestingly, pp125fak activation decreased dramatically in HBC cells cultured for two weeks in suspension, suggesting that activation of this kinase is not necessary for longterm growth under anchorageindependent conditions. These results suggest that constitutive activation of pp125fak results in preferential survival of human breast cancer cells under anchorageindependent conditions but that activation of pp125fak is not the sole mediator of anchorageindependent colony formation.     

Cisplatin-induced reductions in renal functional reserve uncovered by unilateral nephrectomy: an experimental study in the pig

Summary Groups of mature Large White female pigs, approximately 10 months of age, received single intravenous infusions of 1.5, 2 or 2.5 mg/kg body weight (equivalent to90, 120 and 150 mg/m²) cisplatin. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured before and at 4 weeks after cisplatin infusion by renography using [^{99 m}Tc]-DTPA (diethylenetriamminepentaacetic acid and iodohippurate sodium I 131, respectively. The left kidney of each cisplatin-treated animal plus that of four age-matched control pigs was then removed surgically,and GRF and ERPF were measured in the remaining kidney at 4 weekly intervals for up to 24 weeks after unilateral nephrectomy (UN). The pigs treated with cisplatin exhibited no consistent change in either GFR or ERPF at 4 weeks after treatment. A histological evaluation of kidneys from animals treated with 2mg/kg cisplatin that had been removed at UN revealed both tubular and glomerular lesions. The latter consisted of cell proliferation on the parietal surface of the urinary space; damage to the S₁ portion of the proximal convolution was also noted. Following UN there was a pronounced dose-dependent reduction in the functional status of the remaining kidney such that the increase in GRF and ERPF in pigs initially receiving 2.5 mg/kg cisplatin was <50% of that seen in age-matched UN controls. Moreover, the glomerular lesions observed at 4 weeks after cisplatin infusion had apparently progressed to glomerular hyalinisation by 24 weeks after UN. Thus, prior treatment with cisplatin may cause a permanent reduction in renal functional reserve that may be clinically silent until exposure to an additional nephrotoxic insult.This study was supported by the Cancer Research Campaign     

Regulation of estrogen sulfotransferase by estrogen in MCF-7 human mammary cancer cells

Summary The importance of the steroid hormone microenvironment within cells is now recognised in studies on endocrine-related neoplasms such as breast cancer. This focuses attention on ezymes which control the intracellular levels of estradiol-17 (E₂). One such enzyme, estrogen sulfotransferase, which converts E₂ to inactive E₂-3 sulfate, has now been shown to be regulated by estrogen in MCF-7 human mammary cancer cells. Hydroxysteroid sulfotransferase, which sulfurylates the adrenal-derived estrogen 5-androstene-3,17-diol, is also under estrogen control. Evidence is provided which shows that one function of these enzymes may involve elimination of estrogen from the cell following processing of the ligand-charged estrogen receptor (ER).