Interrelationship between O6-alkylguanine-DNA alkyltransferase activity and susceptibility to chloroethylnitrosoureas in several glioma cell lines

Summary In order to study the dynamic relationship in glioma cells between O⁶-alkylguanine-DNA alkyltransferase (AGT) activity and resistance to the cytotoxic effect of chloroethylnitrosoureas (CENUs), we investigated the changes in sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after modulation of AGT activity. In ACNU-resistant rat glioma cell lines (9LR1, 9LR3, and 9LR12) and a human glioma cell (HNG-1), O⁶-methylguanine enhanced cytotoxicity to ACNU following a depletion of AGT activity. But no enhancement of cytotoxicity was seen in an ACNU-sensitive rat glioma cell line (9L). In the 9L and 9LR12 cells, equivalently sublethal doses of ACNU similarly depleted AGT activity but the regeneration rates of this repair protein were different. In the case of a 7-day pretreatment with human recombinant interferon- (HuIFN-), although it could modulate AGT activity in HNG-1 cells, no definite influence on cellular sensitivity to CENUs was observed. However, a 50-day pretreatment with HuIFN- conferred resistance to CENUs on them despite its effect to reduce AGT activity. Thus, diversity was seen in the relation between AGT activity and resistance to CENUs when AGT activity was modulated by HuIFN-. The results of this study suggest that AGT activity is one of factors affecting cellular sensitivity to CENUs but that alternative mechanisms of tolerance may be induced depending upon some environmental effects.     

Interferon-β inhibits proliferation and progression through S phase of the cell cycle in five glioma cell lines

Summary The growth inhibitory effect of IFN- was evaluated in 5 human glioma cell lines (AO2V4, GJC, GJR, NN and NNR) and in normal astrocyte cultures (SC and TM). All 5 glioma cell lines showed an anti-proliferative response to IFN- whereas normal glial cells were non-responsive. IFN- at 10, 100 and 500 U/ml lead to a 30%,70% and 80% relative decrease in cell number after 12 days, respectively in AO2V4 cells. GJC and GJR cell lines also responded significantly to the lowest concentration of IFN- tested and at 500 U/ml the relative cell number decreased 55%. The NN and NNR cells were the least responsive to IFN- with maximum growth inhibition of 30% at 500 U IFN-/ml. Following treatment with IFN-, AO2V4, GJC, GJR and normal astrocytes all expressed mRNA encoding the anti-viral protein, 2-5A synthetase demonstrating that IFN- bound to receptors on all four cell lines and activated signal transduction pathways required for induction of an anti-viral protein. A determination of the relative number of viable cells showed that none of these cells exhibited a significant decrease in cell viability. Since the antiproliferative response to IFN- was not primarily due to cell death, the effect of IFN- on cell cycle progression was evaluated by flow cytometry. All treated glioma cell lines showed a relative increase in proportion of cells in S phase. AO2V4 cells had a 50%–80% increase in the percentage of cells in S phase, whereas GJC, GJR and NNR had percentage increases of 20%–40%. IFN- treatment of normal astrocytes did not significantly alter their cell cycle profile. These data suggest that IFN- exerts its antiproliferative effect on glioma cells by arresting the ordered progression through S phase or decreasing entry into G₂/M phase of the cell cycle.     

Intracellular growth factor metabolism in proliferation of a brain tumor cell line

Brain tumor cells secrete platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-), and through local production of these growth factors, brain tumor cells may stimulate their own proliferation.Previously we have shown that several different clones of canine glioma cells secrete varying amounts of PDGF and TGF- which correlate within vitro cloning efficiency andin vivo tumorigenicity. In this study, intracellular trafficking of PDGF and TGF- was assessed by treatment of each clone with agents preventing vesicular degradation and secretion of growth factors. Clone 2 was more sensitive to these agents (chloroquine and monensin) than clone 5, resulting in retention of intracellular¹²⁵I-PDGF and¹²⁵I-TGF-. Furthermore, exogenous TGF- inhibited DNA-synthesis dramatically in clone 2 (compared with clone 5), presumably by interfering with intracellular growth factor receptor availability. This is supported by the fact that exogenous TGF- increased the number of its receptors on clone 2 cells, whereas surface receptors decreased on clone 5 cells treated with TGF-. These results illustrate the potential for autocrine growth factors to interact with their receptors intracellularly during neoplastic cell proliferation.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Glioma Cell Invasion: Regulation of Metalloproteinase Activity by TGF-β

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF- promotes the migration of LN-18 and LN-229 glioma cells via a process that may involve the upregulation of _V3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-₂ expression. Here, we demonstrate that TGF-₂ induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x_L protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties, TGF- continues to be one of the most promising targets for the experimental therapy of human malignant glioma.     

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.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Characteristics of in vitro antiproliferation activity of human interferon-β

Summary We compared the in vitro antiproliferative activity of highly purified interferon (IFN)- (>10⁷ U protein/mg in antiviral activity) with that of IFNs- and lymphoblastoid, using human cells of malignant and non-malignant origin. IFN- was the least active of three IFNs in suppressing Daudi cell proliferation. Three hematological cells other than Daudi cells cultivated in suspension were insensitive to each of three IFNs. IFN- was more active than IFNs- and lymphoblastoid in suppressing all eight epithelioid cells tested and, particularly with respect to five epithelioid cells sensitive to IFN, IFN- was seven to 49 times as active as IFN-. These results indicate that suppression of cell proliferation by IFN depends not only on the target cell species but also on the IFN species, and emphasize the need for careful selection of the most appropriate IFN species in therapy.We found that the antiproliferative characteristics of the present IFN- preparation were consistent with those reported previously, supporting the idea that IFN- molecules in the present preparation were responsible for suppressing cell proliferation. The antiproliferation activity of our preparation was species-specific but not selective for cells of malignant origin; it was absorbable by IFN-sensitive but not by IFN-insensitive cells; and it was achieved by a cytostatic effect.     

Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1 (IL-1), interferon- (IFN-), vincristine, and etoposide was evaluated on the secretion of transforming growth factor- (TGF-) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1, IFN-, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 and IFN- caused greater inhibition of TGF- secretion compared to treatment with IFN-, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF- secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1, IFN-, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF- and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1, IFN-, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGF1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGF1 neutralizing antibody confirms the correlation between induction of active TGF1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGF1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGF1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Flow cytometric analysis of antineoplastic effects of interferon-α, β and γ labelled with fluorescein isothiocyanate on cultured brain tumors

Summary Antineoplastic effects of interferons (IFNs) on brain tumors have often been reported in the literature, however, so far as we know, there are no reports of the study on the antineoplastic effect of IFNs (, , and ) labelled with fluorescein isothiocyanate (FITC) using flow cytometry (FCM). Three established glioma cell lines and 11 cultured cells of brain tumor from surgical specimens were exposed to IFN-, , and at the concentrations of 10²–10⁵ IU/ml for 24 h, respectively. Using FCM, the viability of the cells was evaluated with fluorescein diacetate stain and the cell cycle was analyzed from the DNA-histogram with propidium iodide stain. Furthermore, FITC-labelled IFN-, and were also contacted with each cell to calculate respective positive cells. The viability decreased about 60% on day 1 and day 3, indicating the effect of IFN- and on U373MG cells and on some cultured glioma cells from surgical materials, whereas, IFN- had no effects. Antineoplastic effect of each IFN well correlated with FITC-positive rates, demonstrating S phase block in the cell cycle. IFN- had no antineoplastic effects, whereas IFN- and showed antineoplastic effects, which fact suggested that IFN- receptor be different from those of IFN- and . The method of FITC-labelling for IFNs with the aid of FCM has the advantages as follows: 1) Antineoplasticity of IFN can be simply evaluated with FCM; 2) It is easy to analyze the action mechanism of IFN; 3) Information on the receptor is obtainable; and 4) Sensitivity can be evaluated prior to administration of IFN, suggesting possibilities of clinical application of this method.     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.     

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CG conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CG-chain in vitro. To test the hypothesis that the Hecate-CG conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CG conjugate and Hecate alone. Cytotoxic effects of the Hecate-CG conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CG conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CG conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC₅₀) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CG for in vivo trials.     

Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon

Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions. Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures. The period required for primary outgrowth ranged from 3 days to 14 days. We established serial propagation with 15/16 of the primary cultures. Sensitivity to HuIFN- was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, netural red). Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech). We observed a broad range of responsiveness to the drug among the 12 cell-strains tested. Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10⁴ IRU/ml of purified HuIFN-. Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups.During propagation of these biopsies, cytopathology suggestive of paramyoxvirus-infection appeared in 4 of the cell-strains. This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance.Our results support the concept that information concerning sensitivity to HuIFN- and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies. Additionally, these results suggest that further clinical studies with interferon should be undertaken to define the parameters which determine successful in vivo application.     

Hematogenous metastases of the human brain – Characteristics of peritumoral brain changes: A review

The brain is an important site of hematogenous metastases from malignanttumors in other organs. The effects on the brain is a combination of tissuedestruction induced by invading tumor cells and reactive alterationsoccurring around the metastases. This review focuses on neuropathologicalchanges around hematogenous metastases of the human brain. The peritumoralbrain parenchyma shows structural and functional changes of theintracerebral microvessels and edema. The endothelial cells of peritumoralmicrovessels express glucose transporter protein (GLUT 1) in the same way asthe normal brain. Reduction in immunostaining to GLUT 1 may occur in themicrovessels located within the metastases. This would indicateabnormalities of the blood-brain barrier in tumor vessels but normal barrierfunction in the peritumoral region. Reactive astrocytes and activatedmicroglial cells are both involved in the process of peritumoral gliosis.Activated glial cells produce numerous biological active compounds includingendothelin-1 which after release from such cells can influence the structureand function of the peritumoral brain tissue. Lesions of oligodendrocytesand edema may be implicated in myelin degeneration. Finally, metastases willinduce axonal and neuronal injuries as indicated by a recent study onexpression of -amyloid precursor protein (APP) in reactive axonalswellings.     

IFN-β Gene Therapy Induces Systemic Antitumor Immunity Against Malignant Glioma

We previously demonstrated that intratumoral administration of liposomes containing the murine interferon beta (IFN-) gene [lip(pSV2muIFN-)] resulted in stronger growth-inhibitory effect on GL261 (H-2^b) mouse glioma inoculated in brains of syngeneic C57BL/6 mice than conventional exogenous IFN- administration, and histologic evaluation revealed the massive infiltration of T lymphocytes (CD8 > CD4) within the residual tumor. The present study was aimed at determining whether such tumor-infiltrating lymphocytes (TIL) have any tumor-specific cytotoxic effects. Intratumoral administration of lip(pSV2muIFN-) resulted in prolonged survival time and a 50% tumor-free incidence in the mice treated. The surviving animals were subsequently re-challenged with either subcutaneous or intracranial injection of GL261 cells, and no tumors were found to develop over a 50-day period. In vivo depletion of CD8, but not CD4 cells decreased the efficacy of lip(pSV2muIFN-). Specific cytotoxic T lymphocytes (CTL) against GL261 cells were generated from both TIL and spleen cells of the mice treated. The results of flow cytometric analysis and antibody blocking test revealed that the bulk CTL lines thus prepared were T cell receptor (TCR) , CD8 T lymphocytes with H-2^b restriction.These findings suggest that, in addition to direct growth-inhibitory effects by the IFN- gene on the tumor cells, activation of systemic cellular immunity may participate in antitumor effects in vivo, despite the fact that central nervous system is generally regarded as an immunologically privileged site.     

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.     

β-Carotene and/or vitamin E as modulators of alkylating agents in SCC-25 human squamous carcinoma cells

Summary Dietary levels of -carotene and vitamin E have been associated with cancer prevention and to a lesser extent, with therapeutic enhancement of cancer treatment. We report on the cytotoxicity of -carotene, vitamin E, and the combination of -carotene and vitamin E in human SCC-25 squamous carcinoma cells under various environmental conditions found in solid tumor masses. -Carotene was selectively cytotoxic toward normally oxygenated cells and was generally more cytotoxic at normal pH than at acidic pH (6.45). Vitamin E was selectively cytotoxic toward normally oxygenated cells following 6 h exposure at normal pH and was generally equally cytotoxic toward normally oxygenated and hypoxic cells under the other conditions tested. -Carotene was an effective modulator of cisplatin (CDDP) cytotoxicity toward SCC-25 cells, whereas vitamin E was not. Both -carotene and vitamin E were effective modulators of melphalan cytotoxicity toward SCC-25 cells. Treatment of SCC-25 cells with -carotene (70 m, 2h) resultec in a reduction in superoxide dismutase activity, in glutathione-S-transferase activity, and in nonprotein sulfhydryl levels in the cells. Exposure to vitamin E or to a combination of -carotene and vitamin E increased the glutathione-S-transferase activity in SCC-25 cells by 40%–45% over the control value. Treatment with -carotene, vitamin E, or canthaxanthin reduced the incorporation of [³H]-thymidine into SCC-25 cells but not that into normal human karotinocytes. The most marked reduction in [³H]-thymidine incorporation into SCC-25 cells occurred following treatment with the combination of -carotene and melphalan. We hope to continue to explore the mechanisms of this effect and to study these combinations in vivo.This work was supported by NIH grant PO1-38 493, by a grant from Bristol-Mycrs-Squibb (Wallingford, Conn.), and by a grant from Cancer Prevention Technologies, Inc. (Basking Ridge, N. J.)