Differences in susceptibility to tumor necrosis factor alpha-induced apoptosis among MCF-7 breast cancer cell variants.

Widespread use of MCF-7 human breast carcinoma cells as a model system for breast cancer has led to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, variations in sensitivity to apoptosis have not yet been described. Tumor necrosis factor alpha (TNF-alpha) has been shown to both induce apoptosis and inhibit proliferation in MCF-7 cells. We observed that TNF-alpha inhibited proliferation in MCF-7 cell variants from three different laboratories (designated M, L, and N). MCF-7 M cells were resistant to TNF-alpha-induced apoptosis, whereas MCF-7 L cells were moderately resistant to the effect of TNF-alpha. A third variant, MCF-7 N, underwent apoptosis when exposed to TNF-alpha. Analysis of the p55 TNF-alpha receptor (TNFR) 1 expression revealed the greatest expression in MCF-7 N cells, whereas the MCF-7 L and M cells expressed 89 and 67% of MCF-7 N cell TNFR1 levels, respectively. Ceramide generation occurred in all three variants in response to TNF-alpha treatment, with MCF-7 N cells expressing the greatest increase. Cleavage of the CPP32/caspase 3 substrate poly(ADP-ribose) was observed in MCF-7 N and L cells as early as 3 and 6 h, respectively, but poly(ADP-ribose) cleavage was not observed in MCF-7 M cells. The delayed protease activation in the L variant may represent the mechanism by which these cells display delayed sensitivity to TNF-a-induced apoptosis. Expression of the Bcl-2, Mcl-1, Bcl-X, Bax, and Bak proteins was analyzed to determine whether the differences in MCF-7 cell sensitivity to apoptosis could be correlated to the differential expression of these proteins. Whereas Bak, Bcl-X, and Mcl-1 levels were identical between variants, the levels of Bcl-2 were 3.5-3.8-fold higher and the levels of Bax were 1.5-1.7-fold lower in the resistant variants (M and L) as compared with those of the sensitive variant (N). Taken together, these results suggest that differences in susceptibility to TNF-alpha-induced apoptosis among MCF-7 breast cancer cell variants may be explained by differences in TNFR expression, ceramide generation, differential expression of the Bcl-2 family of proteins, and protease activation.

Protein kinase C alpha protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia.

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCalpha antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma isoforms, as well as novel PKCepsilon and delta and the atypical zeta isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha isoform plays an important role in sustaining hypoxia-regulated EPO production.

Nuclear protein kinase C activity is decreased in Friend erythroleukemia cells induced to differentiate.

Although it is well known that protein kinase C (PKC) is an important signaling molecule in Friend erythroleukemia cells it is not clear what role PKC may play in either regulated or unregulated erythroid cell proliferation and differentiation. The purpose of this study was to test the hypothesis that a decrease in nuclear PKC activity is associated with the induction of differentiation in Friend erythroleukemia cells. The effects of staurosporine, a selective inhibitor of PKC, and the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, an activator of PKC, on Friend cell proliferation and differentiation were examined. Neither the inhibitor nor the activator of PKC affected proliferation at 96 h as measured by [3H]thymidine incorporation, but both compounds inhibited cell differentiation. In addition, nuclear PKC activity was highest in untreated and in tumor promoter-treated cells that were not differentiated, and it was lowest in cells induced to differentiate with hexamethylene bisacetamide or dimethylsulfoxide. It is concluded that nuclear PKC activity is essential for Friend erythroleukemia cell proliferation, and that a decrease in enzyme activity within the nucleus is associated with differentiation.

Possible involvement of phospholipase activation in erythroid progenitor cell proliferation.

The possible role of phospholipase activation in erythroid progenitor (colony-forming units erythroid; CFU-E) cell proliferation was investigated using a variety of inhibitors of phospholipase activity. Quinacrine and alpha-p-dibromoacetophenone (BPB), both nonselective phospholipase inhibitors, were tested for their effects on CFU-E-derived colony formation. Both drugs significantly inhibited colony formation in the presence or absence of added erythropoietin (Epo). For quinacrine, the concentration causing 50% inhibition (IC50) was 1 microM, whereas for BPB the IC50 was 25 microM. A more selective inhibitor of phospholipase A2, 7,7-dimethyleicosadienoic acid (DEDA), was also tested and the IC50 was 250 microM in the presence of Epo. The addition of phospholipase A or C did not significantly affect CFU-E colony formation, although arachidonic acid increased CFU-E formation as did 12-hydroperoxyeicosatetraenoic acid (12-HPETE), whereas 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and 15-HPETE had no effect. These findings suggest an important role for phospholipase activation in erythroid progenitor cell proliferation.

The neurotrophins and their receptors: structure, function, and neuropathology.

The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

Interleukin-3 or erythropoietin induced nuclear localization of protein kinase C beta isoforms in hematopoietic target cells.

Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effect of both interleukin-3 (IL-3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this study was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factors, IL-3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SUt.EP cell line, a non-transformed hematopoietic cell line that requires IL-3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the alpha, beta I, beta II and gamma isoforms of PKC, we have identified beta I and beta II as the predominant nuclear isoforms in target cells that proliferate in response to IL-3 or EPO.

Changes in redox affect the activity of erythropoietin RNA binding protein.

We have previously identified a cytosolic protein, erythropoietin RNA binding protein (ERBP), which is up-regulated in certain tissues in response to hypoxia. To further characterize the interaction of ERBP and erythropoietin (EPO) mRNA, we have examined the role of reduction-oxidation in the EPO mRNA binding mechanism of ERBP isolated from human hepatoma cells (Hep3B). Reducing agents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) increased ERBP binding activity in a concentration-dependent manner, whereas the oxidizing agent, diamide, abolished ERBP binding activity. In addition, treatment of Hep3B cell lysates with the irreversible sulfhydryl alkylating agent N-ethylmaleimide resulted in inhibition of the EPO mRNA-ERBP complex. Taken together, these findings suggest that sulfhydryl groups may play a role in vivo in the regulation of EPO production through the modulation of ERBP binding activity.

Inhibitory effects of zidovudine in erythroid progenitor cells. Reversal with a combination of erythropoietin and interleukin-3.

To investigate the mechanisms that may be involved in zidovudine (AZT)-induced hematopoietic toxicity, spleen cells isolated from phenylhydrazine-treated anemic mice or murine bone marrow erythroid progenitor cells were treated with AZT (1-10 microM) for 24 hr. A concentration-dependent inhibition of the binding of 125I-labeled erythropoietin (Epo) was observed, suggesting down-regulation of Epo receptors. To determine if this effect is due to modulation of the levels of Epo receptor mRNA and to assess the effect of AZT on the expression of protooncogenes, mRNA levels were monitored by the slot blot hybridization technique. AZT caused a concentration-dependent inhibition in the levels of the mRNA of Epo receptors and c-fos, whereas the level of c-myc mRNA was unaffected. AZT also inhibited protein kinase C (PKC) activity in a concentration- and time-dependent manner, causing 50% inhibition at 10 microM within 3 hr. Simultaneous addition of Epo or interleukin-3 (IL-3) partially reversed the inhibitory effects of AZT on the levels of the mRNAs and on PKC activity; however, a combination of Epo and IL-3 was significantly more effective. These studies demonstrate that (i) AZT-induced down-regulation of Epo receptors and c-fos expression coupled with inhibition of Epo receptor-mediated signal transduction through PKC are significant contributory factors to AZT-induced erythroid toxicity, and (ii) these inhibitory effects can be overcome by treatment with a combination of Epo and IL-3.

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase.

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G0/G1 phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15-60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells.

The molecular details of hypoxia-induced cellular responses have been difficult to identify since there is as yet no known oxygen receptor. We used cDNA microarray technology to extend our studies pertaining to these molecular details in human hepatocellular carcinoma (Hep3B) cells that produce erythropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin precursor and glycogen synthase kinase-3beta (GSK-3beta) were markedly stimulated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low oxygen responses in Hep3B cells. ILK is a serine, threonine protein kinase that interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosphatidylinositol 3-kinase dependent manner and can phosphorylate protein kinase B (PKB/AKT) and GSK-3beta. As a result of these and other activities ILK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated in the Wnt pathway and as a critical target in PTEN-dependent tumor therapies. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatment were elongation factor-1alpha, glycyl-tRNA synthetase, and laminin receptor protein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis.

Differences in protein kinase C and estrogen receptor alpha, beta expression and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants.

Widespread use of MCF-7 human breast cancer cells as a model system for breast cancer has lead to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, differences in sensitivity to apoptosis have just begun to be described. Based on the possible differences in apoptotic sensitivity that may arise due to the existence of MCF-7 cell variants, we determined the relative sensitivity of MCF-7 cell variants from three established laboratories (designated M, L and N) to known inducers of apoptosis. Consistent with our previous studies we demonstrate that differences exist among these variants in regards to tumor necrosis factor alpha (TNF)-induced cell death and inhibition of proliferation in a dose-dependent manner. To establish if the difference in apoptotic susceptibility was specific to TNF, the three MCF-7 cell variants were tested for their response to other known inducers of apoptosis: okadaic acid, staurosporine and 4-hydroxy-tamoxifen. Viability and DNA fragmentation analysis revealed a similar pattern of resistance to apoptosis by all agents in the MCF-7 M variant. The MCF-7 L variant was resistant to okadaic acid and 4-hydroxy-tamoxifen but not staurosporine. In contrast, MCF-7 N cells were sensitive to induction of apoptosis by all agents. The role of both protein kinase C (PKC) and estrogen signaling in the regulation of cell survival prompted investigation of these pathways as a mechanism for differential sensitivity of MCF-7 cell variants to apoptosis. While both estrogen receptor alpha (ERalpha) and ERbeta were expressed in MCF-7 M and N cells, the absence of ERbeta in MCF-7 L cells correlated with decreased estrogen responsiveness of the L variant. Variations in estrogenic responsiveness and PKC isoform expression may account for the enhanced susceptibility of both the L and N variants to staurosporine.

Dexniguldipine hydrochloride, a protein-kinase-C-specific inhibitor, affects the cell cycle, differentiation, P-glycoprotein levels, and nuclear protein phosphorylation in Friend erythroleukemia cells.

Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance. Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity. Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation. P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance. At 2.5 microM, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase. DNA synthesis was decreased in cells exposed to DNIG for 20 h. Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle. Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug. HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation. Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein. Western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC alpha. Finally, levels of P-glycoprotein were decreased in the presence of this drug. Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation. Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance. The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.

Diagnostic use of CFU-E formation from peripheral blood in polycythemia vera.

Peripheral blood mononuclear cells from five patients with polycythemia vera (P. vera) and three with other polycythemias were cultured in a methylcellulose system. Colony-forming unit-erythroid (CFU-E) colonies appeared after seven days in the absence of added erythropoietin (Ep) in all P. vera cultures. A pattern of growth similar to the one seen for P. vera patients occurred in the culture from a patient in whom that disease was suspected. In the cultures from two of the patients with other polycythemias, erythroid colonies did not appear even in the presence of Ep. These findings emphasize the potential value of culturing peripheral blood for CFU-E colonies in diagnosing polycythemia vera.

Oestrogen-mediated suppression of tumour necrosis factor alpha-induced apoptosis in MCF-7 cells: subversion of Bcl-2 by anti-oestrogens.

In oestrogen receptor (ER)-positive breast carcinoma cells, 17beta-oestradiol suppresses a dose-dependent induction of cell death by tumour necrosis factor alpha (TNF). The ability of oestrogens to promote cell survival in ER-positive breast carcinoma cells is linked to a coordinate increase in Bcl-2 expression, an effect that is blocked with the pure anti-oestrogen ICI 182,780. The role of Bcl-2 in MCF-7 cell survival was confirmed by stable overexpression of Bcl-2 which resulted in suppression of apoptosis induced by doxorubicin (DOX), paclitaxel (TAX) and TNF as compared to vector-control cells. The pure anti-oestrogen ICI 182,780 in combination with TNF, DOX or TAX potentiated apoptosis in vector-transfected cells. Interestingly, pre-treatment with ICI 182,780 markedly enhanced chemotherapeutic drug- or TNF-induced apoptosis in Bcl-2 expressing cells, an effect that was correlated with ICI 182,780 induced activation of c-Jun N-terminal kinase. Our results suggest that the effects of oestrogens/anti-oestrogens on the regulation of apoptosis may involve coordinate activation of signalling events and Bcl-2 expression.

NF-kappa B-mediated chemoresistance in breast cancer cells.

BACKGROUND: Nuclear factor-kappa B (NF-kappa B) is a known survival pathway, and it may explain differential sensitivity to tumor necrosis factor-alpha (TNF-alpha) and chemotherapeutic-induced apoptosis in apoptotically sensitive (APO+) and apoptotically resistant (APO-) Michigan Cancer Foundation-7 breast cancer cells. METHODS: Crystal violet viability and luciferase reporter gene assays were used to determine the inhibitory concentration of viability at 50% (IC(50)) and the inhibitory concentration of activity at 50% (EC(50)) values in APO- and APO+ cells with the selective NF-kappa B inhibitor, BAY 11-7082 (BAY). The apoptotic reporter assay was used to determine the effects of the transfection of the inhibitory kappa B-dominant negative (I kappa B-DN) construct in conjunction with TNF, paclitaxel, or doxorubicin treatments in these cells. RESULTS: The concentrations at which 50% of cell viability is inhibited (IC(50)) and at which 50% of NF-kappa B activity is inhibited (EC(50)) for BAY in APO- and APO+ cells were 95.24 micromol/L and 1.53 micromol/L, respectively, and 7.62 micromol/L and 2.64 micromol/L, respectively. The IC(50) and the EC(50) values were equivalent for the APO+ cells (P =.665), but not for the APO- cells (P =.025). I kappa B-DN--transfection alone, or with TNF, doxorubicin, or paclitaxel treatments resulted in cell death of both APO- and APO+ cells as compared with vector-control; however, greater cytotoxicity was seen in the APO+ cells. Direct comparison of the APO+ cells versus the APO- cells revealed that these differences were significant (P =.05). CONCLUSIONS: Pharmacologic or molecular inhibition of the NF-kappa B pathway blocked cell survival in MCF-7 APO+ cells, while only molecular inhibition induced cytotoxicity in the APO- cells. Selective manipulation of the NF-kappa B pathway in combination with standard chemotherapeutic agents may lead to an increased potency and efficacy of these agents.

Anti-HIV drugs: comparative toxicities in murine fetal liver and bone marrow erythroid progenitor cells.

The toxicity of anti-HIV drugs, 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine (d4T) and ribavirin was studied in vitro in murine fetal liver cells (FLC) and in bone marrow cells. These studies indicate that d4T is the least toxic drug and ribavirin is the most toxic agent in both models. However, the murine FLC system was found to be a more sensitive model for the assessment of toxicity of anti-HIV agents towards erythroid progenitor cells as indicated by the IC50 values.

PH20: a novel tumor marker for laryngeal cancer.

OBJECTIVE: To determine whether levels of PH-20, a hyaluronidase similar to that found in human sperm, are elevated in laryngeal cancer tissue. DESIGN: In this case-control study. reverse transcription polymerase chain reaction was used to measure levels of PH-20 messenger RNA in tissue taken from laryngectomy specimens. SETTING: A university medical center. PATIENTS: We compared tissue samples taken from 11 patients with laryngeal cancer, and from 2 metastatic lymph nodes, with samples of normal, healthy laryngeal tissue and prostate cancer tissue (positive control). MAIN OUTCOME MEASURE: PH-20 complementary DNA expression as quantified by densitometric analysis. RESULTS: Expression of PH-20 was significantly higher in nonirradiated laryngeal cancer specimens than in normal laryngeal tissue (P<.01). Metastatic lymph nodes also had higher levels of PH-20 expression than did primary laryngeal cancer tissue (P = .11) and normal laryngeal tissue (P<.01). Irradiated laryngeal cancer specimens had PH-20 levels comparable to normal. CONCLUSIONS: We report the first data on PH-20 expression in laryngeal cancer tissue. PH-20 expression is significantly elevated in primary laryngeal cancer tissue and seems to be even higher in metastatic lesions compared with normal laryngeal tissue. PH-20 may be a useful tumor marker and prognostic tool for laryngeal cancer.