Biomedical subjects

S J Berger

Publications and source records attributed to S J Berger.

At least 19 recordsLinked to original sources

PubMed2001

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells.

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.

Antineoplastic Agents↗

PubMed2001

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Chromatography, Liquid↗

PubMed2001

High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts.

High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.

Bacterial Proteins↗

PubMed2001

Phase II and pharmacokinetic/pharmacodynamic trial of sequential topoisomerase I and II inhibition with topotecan and etoposide in advanced non-small-cell lung cancer.

PURPOSE: In vitro and in vivo preclinical models have demonstrated synergistic activity when topoisomerase I and II inhibitors are administered sequentially. Topoisomerase I inhibitors increase topoisomerase II levels and increase cell kill induced by topoisomerase II poisons. We evaluated this hypothesis in a cohort of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: A group of 19 patients with advanced NSCLC (70% adenocarcinoma) received topotecan at a dose of 0.85 mg/m2 per day as a continuous 72-h infusion from days 1 to 3. Etoposide was administered orally at a dose of 100 mg twice daily for 3 days on days 7-9 (schedule and dose derived from prior phase I trials). Total and lactone topotecan concentrations were measured at the end of the 72-h infusion. Blood samples were obtained immediately after each 72-h topotecan infusion in order to measure the mutational frequency at the hypoxanthine phosphoribosyl transferase (HPRT) locus in peripheral lymphocytes. RESULTS: A total of 55 cycles were administered. Toxicity was mainly hematologic with grade 4 neutropenia occurring in 7% of courses. Only one partial response and two stable diseases were observed. The 1-year survival rate was 33%. There was a statistically significant difference between steady-state lactone concentrations between cycle 1 and cycle 2 with decreasing concentrations with cycle 2 (P = 0.02). This was explained by a statistically significant increase in the clearance of topotecan lactone during cycle 2 (P = 0.03). Total but not lactone concentrations correlated with nadir WBC, ANC and platelet levels. Steady-state plasma lactone levels correlated with the mutational frequency at the HPRT locus (P = 0.06). In the one patient with a partial response a sixfold increase in HPRT mutational frequency was observed, which was not seen in patients with progressive disease. CONCLUSION: The combination of topotecan and etoposide in this schedule of administration has minimal activity in adenocarcinoma of the lung. This lack of activity may be due to the delay in administration of etoposide after the topotecan as studies have shown that the compensatory increase in topoisomerase II levels after treatment with topoisomerase I inhibitors is shortlived (<24 h). The HPRT mutational frequency results suggest that the lack of clinical response may be associated with failure to achieve sufficient cytotoxic dose as indicated by a lack of increase in mutational frequency in those patients with progressive disease. HPRT mutational frequency may correlate with plasma steady-state topotecan lactone levels. Future studies should be directed toward earlier administration of topoisomerase II inhibitors after topoisomerase I inhibition.

Adult↗

PubMed2001

Sequential tumor biopsies in early phase clinical trials of anticancer agents for pharmacodynamic evaluation.

PURPOSE: In the setting of target-based anticancer drug development, it is critical to establish that the observed preclinical activity can be attributed to modulation of the intended target in early phase trials in human subjects. This paradigm of target modulation allows us to determine a Phase II or III dose (optimal biochemical/biological modulatory dose) that may not necessarily be the maximum tolerated dose. A major obstacle to target-based (often cytostatic) drug development has been obtaining relevant tumor tissue during clinical trials of these novel agents for laboratory analysis of the putative marker of drug effect. EXPERIMENTAL DESIGN: From 1989 to present, we have completed seven clinical trials in which the end point was a biochemical or biological modulatory dose in human tumor tissues (not surrogate tissue). Eligibility enrollment required that patients have a biopsiable lesion either with computerized tomography (CT) guidance or direct visualization and consent to sequential (pre and posttreatment) biopsies. RESULTS: A total of 192 biopsies were performed in 107 patients. All but 8 patients had sequential pre and posttreatment biopsies. Seventy-eight (73%) of the 107 patients had liver lesion biopsies. In eight patients, either one or both biopsies contained insufficient viable tumor tissue or no tumor tissue at all for analysis. Of a total of 99 patients in whom we attempted to obtain paired biopsies, a total of 87 (88%) were successful. Reasons for failure included patient refusal for a second biopsy (n = 2), vasovagal reaction with first biopsy precluding a second biopsy (n = 1), subcapsular hepatic bleeding (n = 1), and most commonly obtaining necrotic tumor, fibrous, or normal tissue in one of the two sequential biopsies (n = 8). CONCLUSIONS: This is the first and largest reported series demonstrating that with adequate precautions and experience, sequential tumor biopsies are feasible and safe during early phase clinical trials.

Animals↗

PubMed2000

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Animals↗

PubMed2000

Capillary isoelectric focusing of yeast cells.

In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (approximately 4-microm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-microm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/microL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.

Isoelectric Focusing↗

PubMed2000

High-efficiency capillary isoelectric focusing of peptides.

Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.

Amino Acid Sequence↗

PubMed2000

Phase I and pharmacologic study of irinotecan administered as a 96-hour infusion weekly to adult cancer patients.

PURPOSE: We conducted a phase I and pharmacologic study of a weekly 96-hour infusion of irinotecan to determine the maximum-tolerated dose, define the toxicity profile, and characterize the clinical pharmacology of irinotecan and its metabolites. PATIENTS AND METHODS: In 26 adult patients with solid tumors, the duration and dose rate of infusion were escalated in new patients until toxicity was observed. RESULTS: In 11 patients who were treated with irinotecan at 12.5 mg/m(2)/d for 4 days weekly for 2 of 3 weeks, dose-limiting grade 3 diarrhea occurred in three patients and grade 3 thrombocytopenia occurred in two patients. The recommended phase II dose is 10 mg/m(2)/d for 4 days given weekly for 2 of 3 weeks. At this dose, the steady-state plasma concentration (Css) of total SN-38 (the active metabolite of irinotecan) was 6.42 +/- 1.10 nmol/L, and the Css of total irinotecan was 28.60 +/- 17.78 nmol/L. No patient experienced grade 3 or 4 neutropenia during any cycle. All other toxicities were mild to moderate. The systemic exposure to SN-38 relative to irinotecan was greater than anticipated, with a molar ratio of the area under the concentration curve (AUC) of SN-38 to irinotecan of 0.24 +/- 0.08. One objective response lasting 12 months in duration was observed in a patient with metastatic colon cancer. CONCLUSION: The recommended phase II dose of irinotecan of 10 mg/m(2)/d for 4 days weekly for 2 of 3 weeks was extremely well tolerated. Further efficacy testing of this pharmacologic strategy of administering intermittent low doses of irinotecan is warranted.

Adult↗

PubMed1999

GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

ADP-Ribosylation Factors↗

PubMed1999

Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation.

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.

6-Aminonicotinamide↗

PubMed1999

Poly(ADP-ribose) polymerase: a guardian of the genome that facilitates DNA repair by protecting against DNA recombination.

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.

Animals↗

PubMed1998

Analysis of recombinant human ADP-ribosylation factors by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry.

Two complementary approaches utilizing reverse-phase high-performance liquid chromatography and liquid chromatography/mass spectrometry were developed to analyze recombinantly produced Group I and Group II human ADP-ribosylation factors (ARFs). We observe that the NH2 termini of Group II ARFs (ARF4 and ARF5) are efficiently processed by removal of the initiating methionine. In contrast, the NH2 termini of Group I ARFs (ARF1 and ARF3), although fully deformylated, undergo only partial methionine cleavage. This result is unexpected as ARFs are canonical substrates for methionine processing in both bacterial and eukaryotic systems, but it may explain the difficulties encountered by many researchers attempting to produce myristoylated ARFs in Escherichia coli. Additionally, we observe that a significant fraction of purified ARF4 contains a modification which we demonstrate to be consistent with mono-glutathionation. Both methionine retention and glutathione modification may impact ARF function and the methods presented here should be employed to determine the quality of recombinant ARFs.

ADP-Ribosylation Factor 1↗

PubMed1997

Hypersensitivity to DNA cross-linking agents associated with up-regulation of glucose-regulated stress protein GRP78.

We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.

6-Aminonicotinamide↗

PubMed1996

Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol.

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.

ADP-Ribosylation Factor 1↗

PubMed1995

Mass-spectrometric analysis of ADP-ribosylation factors from bovine brain: identification and evidence for homogeneous acylation with the C14:0 fatty acid (myristate).

The two proteins from bovine brain previously shown to be required for the guanosine 5'-[gamma-thio]triphosphate-dependent inhibition of a well-characterized intra-Golgi transport assay, termed GGBF and GGBF, have been definitively identified as members of the ADP-ribosylation factor (ARF) family by electrospray MS analysis of the intact proteins, and of their tryptic fragments. Extensive protein-sequence information obtained from this analysis identified GGBF and GGBF as bovine ARF1 and ARF3 respectively. The sequence of bovine ARF3, which had not previously been determined, appears identical to that predicted from the rat and human ARF3 cDNAs. Further analysis of the N-terminal tryptic fragments of both bovine ARFs demonstrates N-terminal acylation solely with the C14:0 fatty acid (myristate). This finding establishes that the previously reported specific-activity difference between ARF1 and ARF3 in the intra-Golgi transport assay is not due to lipid heterogeneity at the N-terminus. This finding also indicates that the heterogeneity of N-terminal fatty-acyl groups previously observed on other myristoylated proteins is not universal.

ADP-Ribosylation Factor 1↗

PubMed1995

Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences.

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.

Animals↗

PubMed1995

Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide.

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.

6-Aminonicotinamide↗