BACKGROUND:GRP78 is one of the stress proteins linked to different functions in the cell. Previous reports have shown opposing functions of GRP78 in relation to drug resistance/sensitivity. In the current study, we examined the role of GRP78 in cisplatin-treated A549 cells.MATERIALS AND METHODS:GRP78 was over-expressed in A549 cells with 2-deoxyglucose (2-dG) or tunicamycin (TM) treatments for 48 h and subsequently exposed to cisplatin for 2 h. Viability of these cells was determined at 0, 12, 24, 36 and 48 h afterwards.RESULTS:We showed that A549 cells are hypersensitized to cisplatin following a transient GRP78 up-regulation. This hypersensitization is caused by the activation of JNK pathway and NF-κB, leading to early onset of apoptosis.CONCLUSION:Induction of GRP78 can be used as a potential tool to overcome drug resistance in lung cancer cells.

Background: The specific signaling that occurs between the endoplasmic reticulum (ER) and the nucleus in response to ER stress is known as the unfolded protein response (UPR). Specific induction of GRP78 (glucose-regulated protein of Mr 78 kDa) is an integral component of ER stress and the UPR. We first discovered that the up-regulation of GRP78 is associated with augmented sensitivity/apoptosis of cancer cells to clinically used alkylating/platinating agents. Objectives: To decipher molecular mechanisms that associate induction of the UPR/GRP78 with augmented sensitivity/apoptosis to cisplatin. Materials and Methods: A549 cells were exposed to 2-deoxyglucose (2dG) to induce the UPR/GRP78, followed by cisplatin treatment. We used human cDNA microarray containing 42,000 ESTs as well as pathway-specific macroarrays for apoptosis, cell cycle, and MAP kinase signaling pathways containing 100-280 genes and subsequently examined the pertinent transcript levels. The results obtained from these studies were confirmed by examining relevant protein levels and the enzymatic activity. Results: We demonstrate that the induction of UPR/GRP78 alone causes a decrease in the transcript levels of DNA repair genes and DNA damage check point genes, and an increase in the transcript levels of apoptotic genes. Furthermore, we show that cisplatin treatment after the induction of UPR/GRP78 is facilitating the mitochondria-mediated apoptotic cascades through the initial activation of caspase-2 and down-regulation of genes involved in DNA repair. Conclusions: Our study will shed new insight as to the increased understanding of the mechanisms of the UPR/GRP78 modulation of molecular and cellular responses to cisplatin that will allow strategies for transferring bench side results to the bed.

We previously demonstrated an excellent association between the induction of GRP78 and hypersensitivity to alkylating/platinating agents. However, the mechanisms of this association are still not clear. GRP78 is a strong Ca++ binding chaperone protein residing in the endoplasmic reticulum (ER). Thus, GRP78 induction can potentially alter Ca++ homeostasis in the ER resulting in alteration in the Ca++‐dependent signaling pathways. We verified this hypothesis by examining the transcript levels using human cDNA microarrays and pathway specific macroarrays. A549 human lung cancer cells were exposed to 10 mM 2‐deoxyglucose or 25 nM thapsigargin or 250 nM tunicamycin for 48 h to induce GRP78, followed by 60 μM cisplatin for 2 h. We show that GRP78 induction alone causes a decrease in the transcript levels of DNA repair genes, DNA damage check point genes and an increase in that of apoptotic genes. Further, cisplatin treatment after GRP78 induction is augmenting the mitochondria‐mediated apoptotic cascades through the activation of caspase‐2 and down‐regulation of many genes involved in DNA repair. These results were further confirmed by examining relevant protein levels and enzyme activity. Our study will hence reveal insight into the molecular mechanisms of association between GRP78 induction and hypersensitivity to cisplatin that will ultimately allow strategies for transferring bench side results to the bed.

Resistance to etoposide (VP-16), amsacrine (mAMSA), and doxorubicin (Adriamycin) was studied in two Chinese hamster cell lines primarily selected for resistance to the epipodophyllotoxin. Both lines demonstrated profound resistance to VP-16, and mAMSA stimulated DNA breakage. However, the resistance to mAMSA cytotoxicity in both lines was less than expected from the level of resistance to the effects of topoisomerase II inhibition. Similarly, resistance to the cytotoxicity of high VP-16 concentrations in one of the lines was less than expected from the resistance to inhibition of topoisomerase II. An analysis of the relation of DNA breaks to drug cytotoxicity suggests that cross-resistance to mAMSA was mainly conferred through loss of mAMSA-stimulated, topoisomerase II-mediated DNA breaks. This mechanism also contrib uted towards reduced VP-16 cytotoxicity. However, our studies suggest that additional mechanisms, independent of resistance to VP-16-mediated topoisomerase II effects, greatly increased the resistance to this agent. Resistance to VP-16 cytotoxicity, not dependent on resistance to drugmediated DNA cleavage, could be overcome at high drug concentrations in one of the resistant lines and might be responsible for the greater relative resistance to VP-16 than to mAMSA. These findings suggest the presence of two distinct mechanisms of resistance to VP-16 cytotoxicity, one presumably mediated by topoisomerase II and dependent on resist ance to drug-mediated DNA scission, and a second mechanism independ ent of the effects of the drug on topoisomerase II.

Glucose-regulated protein of M(r) 78kDa (GRP78) is a resident protein of endoplasmic reticulum (ER). We have previously shown that the cells become resistant to topoisomerase II alpha (topo II alpha) targeted cancer chemotherapeutic drug such as etoposide (VP-16) when GRP78 is up-regulated by various means. Up-regulation of GRP78 in V79 Chinese hamster cell lines was achieved by treating the cells with NAD antagonist 6-aminonicotinamide (6AN), inhibitor of glucose metabolism such as 2-deoxyglucose (2dG). Further, up-regulation of GRP78 was also observed in V79-derived cell lines which are deficient in poly(ADP-ribose) polymerase (PARP1) metabolism. However, mechanisms of association of GRP78 up-regulation and resistance to VP-16 remained obscured under the conditions outlined above. In the manuscript, using various methods, we demonstrate, for the first time, that up-regulation of GRP78, using approaches depicted above, causes down-regulation of topo II alpha and its activity. We have also discussed the clinical implications of our findings.

We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.

Cell lines deficient in poly(ADP-ribose) synthesis due to enzyme defi ciency (ADPRT54 and ADPRT3S1) or substrate deficiency (N2, N3, and N4) are resistant to topoisomerase H-directed agents, including etoposide (VP-16), Ar-(4-(9-acridinylamino)-3-methoxyphenyl)methanesulfonamide, and Adriamycin, relative to the effect of these agents on parental V79 Chinese hamster cells. Resistance is stable in the ADPRT54 and ADPRT351 cell lines, whereas resistance in the N2, N3, and N4 cell lines occurs when the cells are grown in nicotinamide-deficient medium to produce a state of NAD deficiency. However, sensitivity to VP-16 reverts to normal when cellular NAO levels return to control levels during growth in nicotinamide-containing complete medium. Poly(ADP-ribose) polymer- ase-deficient cell lines show constitutively increased levels of a protein at I/, 78,000 on Coomassie blue-stained, sodium dodecyl sulfate-polyacryl- amide gels that was subsequently confirmed with monoclonal antibodies to be U, 78,000 glucose-regulated stress protein (GRP78). Similarly, N2, N3, and N4 cells show induction of GRP78 under nicotinamide-deficient conditions. Induction of GRP78 is associated with elevated levels of GRP78 mRNA and appears to be regulated at the transcriptional level. When N3 cells with deficiency of poly(ADP-ribose) synthesis due to NAD deficiency are shifted to complete, nicotinamide-containing medium, they restore their NAD content, undergo a decrease in GRP78 levels, and regain sensitivity to VP-16. When V79 cells are shifted to nicotinamide- deficient medium they undergo a reduction in NAD content, followed by a progressive elevation in GRP78 levels, and they subsequently become increasingly resistant to VP-16. These studies demonstrate a clear associ ation between deficiency of the NAD-poly(ADP-ribose) synthesis system, induction of GRP78 synthesis, and resistance to VP-16.

ADPRT 54 and ADPRT 351, are Chinese hamster, V79, derived mutant cell lines, selected for their marked deficiency in poly(ADP-ribose) polymerase activity (1). These cells were employed to analyze the effect of poly(ADP-ribose) polymerase deficiency on responsiveness to a series of DNA damaging agents including monofunctional and bifunctional alkylating agents, topoisomerase I and II inhibitors and X- and UV-irradiation (1-4). In contrast to previous studies, the use of these poly(ADP-ribose) polymerase deficient, mutant cell lines and comparison to events in normal, parental V79 cells allows for studies of the role of poly(ADP-ribose) polymerase without the use of enzyme inhibitors which could have multiple nonspecific effects.

To get an idea about the response of a living system, exposed to gradually increasing doses of a mutagen for several generations, a population of V-79 Chinese hamster cells was exposed repeatedly to gradually increasing doses of UV radiation. Each dose was followed by a variable period of growth for at least ten generations. After treatment the cells were not mutable by UV radiation, though MNNG was capable of producing mutations with the same efficiency as in the untreated cells. In terms of viability, the treated cells behaved exactly as the untreated ones for both UV and MNNG. The observed behaviour of the treated cells was found to be stable for during the 50 passages studied.