Christine M. Berns

Effect of Ionizing Radiation on AP-1 Binding Activity and Basic Fibroblast Growth Factor Gene Expression in Drug-sensitive Human Breast Carcinoma MCF-7 and Multidrug-resistant MCF-7/ADR Cells

We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress. Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein. H-7 (60 μg/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this observation, overexpression of PKCα induced bFGF gene expression in MCF-7 cells. Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors. Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines. Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of αB-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10−1 isosurvival was 1.7. Expression of αB-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected αB-crystallin on thermoresistance and thermotolerance. Cells stably transfected with αB-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive αB-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of αB-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected αB-crystallin can contribute to increased thermoresistance.

Comparison of tumor growth between HSP25‐ and HSP27‐transfected murine L929 cells in nude mice

We have developed a novel system for examining the possible contribution of small heat shock proteins (hsp) to tumor growth. L929 fibrosarcoma cells, which do not express significant levels of endogenous hsp25, were stably transfected with either murine hsp25 or human hsp27. Both transfected genes were over‐expressed and the respective proteins were phosphorylated in L929 cells. L929 cells transfected with hsp25 exhibited enhanced tumor growth compared to control transfected L929 cells upon s.c. injection into nude mice. In contrast, cells transfected with hsp27 exhibited delayed tumor progression in comparison to controls. Although these 2 heat shock genes and respective proteins are structurally very similar, they apparently exhibit distinct effects on tumor growth in this system. Int. J. Cancer 72:871–877, 1997.

DIFFERENTIAL EFFECT OF GLUCOSE DEPRIVATION ON MAPK ACTIVATION IN DRUG SENSITIVE HUMAN BREAST CARCINOMA MCF-7 AND MULTIDRUG RESISTANT MCF-7/ADR CELLS

We have investigated the effect of glucose deprivation treatment on the activation of mitogen activated protein kinases (MAPKs) in the drug-sensitive human breast carcinoma cells (MCF-7) and its drug resistant variant (MCF-7/ADR) cells. Western blots and in-gel kinase assays showed that glucose free medium was a strong stimulus for the activation of MAPK in MCF-7/ADR cells. No activation was seen in MCF-7 cells. MAPK was activated within 3 min of being in glucose free medium and it remained activated for over 1 h in MCF-7/ADR cells. After being returned to complete medium, 1 h was required for the MAPK to become deactivated. To investigate whether alternative sources of ATP could inhibit glucose deprivation induced MAPK activation, we added glutamine and glutamate to glucose deprived medium. The addition of glutamine did not reverse glucose deprivation induced MAPK activation in MCF-7/ADR cells. The addition of glutamate, however, decreased the MAPK activation and the length of time of activation. We observed an increase greater than three fold in MEK, Raf, Ras, and PKC activity with glucose deprivation in MCF-7/ADR cells. This suggests that glucose deprivation-induced MAPK activation is mediated through this signal transduction pathway.

Hypoglycemia-induced AP-1 transcription factor and basic fibroblast growth factor gene expression in multidrug resistant human breast carcinoma MCF-7/ADR cells

We investigated the effect of hypoglycemic treatment on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in multidrug resistant human breast carcinoma MCF-7/ADR cells. Northern blot and gel mobility shift assays showed that hypoglycemic treatment induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression. Moreover, transfected cells expressing high levels of abnormal c-Jun protein exhibited a reduction in the bFGF protein levels compared to parental cells. A potent protein kinase C (PKC) inhibitor, H-7 (60 μg/ml) suppressed the stress-induced bFGF gene expression. Our study also demonstrated that H-7 did not facilitate the decay of bFGF mRNA. Thus, the suppression of bFGF gene expression by treatment with H-7 was due to the effect of the drug on the synthesis of bFGF mRNA rather than the stability of bFGF mRNA. Our data suggest that hypoglycemia-induced bFGF gene expression is mediated through the activation of PKC and the AP-1 transcription factors. (Mol Cell Biochem 155: 163–171, 1996)

Elevated levels of ERK2 in human breast carcinoma MCF‐7 cells transfected with protein kinase Cα

Abstract. We investigated the effect of elevated levels of protein kinase Cα (PKCα) on cell proliferation in human breast carcinoma cells (MCF‐7). MCF‐7 cells transfected with either the pSV2M(2)6 vector without the insert (MCF‐7/Vector) or containing a full length cDNA encoding PKCα (MCF‐7/PKCα) were compared. MCF‐7/PKCα cells were found to have an increased proliferative rate with a doubling time of 15 h as compared to 42 h for MCF‐7/Vector cells. Flow cytometry illustrated a greater percentage of MCF‐7/PKCα cells in the S phase of the cell cycle. Western and Northern blot analyses demonstrated an increase in extracellular regulated protein kinase 2 (ERK2) gene expression in MCF‐7/PKCα cells but no alteration of this gene expression in MCF‐7/Vector cells. These results suggested that the elevated level of ERK2 which is also known as mitogen activated protein kinase is probably involved in the increase in MCF‐7/PKCα cell proliferation.

Effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on HSP70 and HSP28 gene expression and thermotolerance development in human colon carcinoma cells

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C (PKC) inhibitor, on the development of thermotolerance and expression of heat shock genes (HSP70 and HSP28) was investigated in human colon carcinoma HT-29 cells. After acute heating at 45 degrees for 15 min, cells became resistant to a challenge heat shock. The development of thermotolerance was suppressed by adding H-7 after heat shock. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 6 hr. H-7 suppressed the accumulation of HSP70 and HSP28 mRNA as well as their protein synthesis, and the level of suppression was concentration dependent. However, little effect was observed if the drug was added 1 hr before and during heat shock. These results suggest that PKC is involved in the regulation of heat shock gene expression after acute heat shock.

Examination of the molecular basis for the lack of αB-crystallin expression in L929 cells

We have previously shown that murine L929 cells do not express the small heat shock protein αB-crystallin upon exposure to thermal stress (Mol Cell Biochem 155: 51–60, 1996). In these studies, we demonstrate that L929 cells also fail to express αB-crystallin upon exposure dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit αB-crystallin expression under identical conditions. Mobility shift assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an αB-crystallin heat shock element (HSE) oligomeric sequence in total cellular extracts from L929 cells. Transient transfection of a plasmid containing the αB-crystallin promoter linked to a CAT reporter gene exhibited heat-inducible expression in L929 cells. In addition, L929 cells stably transfected with a plasmid containing the complete αB-crystallin gene showed expression of this gene following heat shock. The presence of the endogenous αB-crystallin gene was detected by Southern blot hybridization of genomic L929 DNA, and sequence analysis revealed identical nucleotide structure to published murine sequences throughout the entire promoter. Treatment of L929 cells with 5-azacytidine enabled heat-inducible expression of αB-crystallin from the endogenous gene, however, methylation of the putative heat shock element (HSE) and flanking promoter sequences of L929 cell genomic DNA was not detected. In vivo genomic footprinting demonstrated constitutive binding to the endogenous HSE of the αB-crystallin promoter in L929, L929/αB-crystallin transfectant cells, and Swiss 3T3 cells during unstressed and heat stressed conditions. Therefore, the genomic αB-crystallin HSE region in L929 cells appears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the expression of αB-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the αB-crystallin gene loci inactive through methylation, thus providing a unique system by which to study the function of transfected small heat shock proteins.

Differential effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on αB-crystallin and hsp70 gene expression in murine cell lines

We studied the effect of isoquinolinesulfonamide derivatives (H-7, H-8, and HA1004) on the expression of two heat shock genes (alpha beta-crystallin and hsp70) in NIH 3T3 and Swiss 3T3 cells after heat shock at 45 degrees for 10 min. Western blots and northern blots showed that H-7 effectively suppressed the accumulation of HSP70 and alpha B-crystallin mRNA as well as the synthesis of their proteins. The degree of suppression was dependent upon the concentration of the drug. Moreover, the expression of the hsp genes was differentially suppressed by H-7. The expression of the alpha B-crystallin gene was more effectively inhibited than that of the hsp70 gene by H-7. Nuclear run-on assay demonstrates that this difference was due to the differential effect of H-7 on the elongation of transcription of different hsp genes.

Transfection of human HSP27 in rodent cells: Absence of compensatory regulation between small heat shock proteins

Abstract 1. 1. We examined rodent cells transfected with an expression plasmid encoding a human small heat shock protein for possible compensatory expression of endogenous heat shock genes. For these investigations, human hsp 27 was transfected into CHO cells which express endogenous HSP25. 2. 2. Both endogenous HSP25 and transfected HSP27 were expressed and multiple phosphorylated isoforms were detected upon exposure to thermal stress. 3. 3. Levels of endogenous HSP70 and HSP25 did not appear to be altered by expression of the heterologous heat shock protein. 4. 4. These results suggest that compensatory interactions are not exhibited in the expression of the heat shock genes examined, and that independent regulation may exist not only between the large and small heat shock proteins, but also between individual small heat shock proteins as well.