We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

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 micrograms/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.

We investigated the effect of elevated levels of protein kinase C alpha (PKC alpha) 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 PKC alpha (MCF-7/PKC alpha) were compared. MCF-7/PKC alpha 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/PKC alpha 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/PKC alpha 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/PKC alpha cell proliferation.

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. © 1997 Wiley‐Liss, Inc.

We investigated the role of alpha B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45 degrees C induced thermotolerance in these cells to a heat challenge at 45 degrees C administered 24 h later. The thermotolerance ratio at 10(-1) isosurvival was 1.7. Expression of alpha B-crystallin gene was not detected during the 24 h incubation at 37 degrees 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 alpha B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with alpha B-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 alpha B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of alpha B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected alpha B-crystallin can contribute to increased thermoresistance.