This chapter focuses on development of resistance to anticancer agents. In recent years there has been great progress made in identifying new mechanisms of resistance. From these studies, it has become clear that genomic instability and the tumor microenvironment strongly influence the development of resistance and play a major role in the evolution of resistance. A large number of scientific studies have focused on the identification and characterization of mechanisms of drug resistance using tissue culture-based models. In general, the mechanisms of microenvironment-related drug resistance can be subdivided into those that lead to reduced drug effect and those mechanisms that provide increased tolerance to damage. In a study with small cell lung cancer (SCLC) cells, extracellular matrix (ECM) proteins were found to protect the cells from chemotherapy induced apoptosis. While the focus on these highly resistant cell lines facilitated the elucidation of the mechanisms behind high level drug resistance, it is possible that the initial lines in which resistance was two-to-fourfold, relative to the original parent cell line, contained the secrets to understanding clinically relevant cancer drug resistance. Ultimately, the identification of the cellular factors that mediate this form of resistance may provide novel strategies for circumventing both intrinsic and acquired drug resistance. It was clear almost from the beginning that multidrug resistance in vitro and in vivo involved multiple mechanisms acting in concert to modulate chemo-sensitivity, although often this was not readily appreciated.

Drug resistance, either intrinsic or acquired, remains one of the primary causes of failure for cancer chemotherapy. This chapter focuses on alterations in the carrier mediated transport systems that are responsible for intrinsic and acquired resistance. As with the antifolate drugs, a single, or a combination of different, mechanism(s) can mediate resistance to nucleoside anticancer agents. Reduced cellular accumulation of anticancer agents is one of the most studied forms of multidrug resistance (MDR). In terms of cellular resistance to anticancer agents, at least 10 family members have been implicated in drug resistance (MRP1-7, MDR1, BCRP, and ABCA2), and four of these have been shown to generate a resistance phenotype in cultured cells. Resistance to several different anticancer agents that are substrates for glutathione-S-transferase (GST) is associated with increases in GST levels (e.g., chlorambucil, melphalan). Interestingly, increased GST levels have been shown to be associated with resistance to non-GST-substrate anticancer agents. Commonly used camptothecins include topotecan and irinotecan. Cell culture based experiments have elucidated several point mutations in the TopoI enzyme, which are responsible for resistance. It should be noted that mutations in TopoI produce resistance to a class of molecules but do not always grant cross-resistance to other TopoI inhibitors. A better understanding of the mechanisms operating at the clinical level is crucial for the development of drugs aimed at circumventing resistance or therapeutic regimens for preventing the emergence of resistance.

Background: Pancreatic adenocarcinoma is an almost universally lethal disease, in large part, due to our inability to detect early-stage disease. Monoclonal antibody PAM4 is reactive with a unique biomarker expressed by >85% of pancreatic adenocarcinomas. In this report, we examined the ability of a PAM4-based immunoassay to detect early-stage disease. Materials and Methods: The PAM4-based immunoassay was used to quantitate antigen in the serum of healthy volunteers (n = 19), patients with known pancreatic adenocarcinoma (n = 68), and patients with a primary diagnosis of chronic pancreatitis (n = 29). Results: Sensitivity for detection of pancreatic adenocarcinoma was 82%, with a false-positive rate of 5% for healthy controls. Patients with advanced disease had significantly higher antigen levels than those with early-stage disease (P < 0.01), with a diagnostic sensitivity of 91%, 86%, and 62% for stage 3/stage 4 advanced disease, stage 2, and stage 1, respectively. We also evaluated chronic pancreatitis sera, finding 38% positive for antigen; however, this was discordant with immunohistochemical findings that suggest the PAM4 antigen is not produced by inflamed pancreatic tissue. Furthermore, several of the serum-positive pancreatitis patients, for whom tissue specimens were available for pathologic interpretation, had evidence of neoplastic precursor lesions. Conclusions: These results suggest the use of the PAM4 serum assay to detect early-stage pancreatic adenocarcinoma and that positive levels of PAM4 antigen are not derived from inflamed pancreatic tissues but rather may provide evidence of subclinical pancreatic neoplasia. Effect: The ability to detect pancreatic adenocarcinoma at an early stage could provide for early therapeutic intervention with potentially improved patient outcomes. Cancer Epidemiol Biomarkers Prev; 19(11); 2786–94. ©2010 AACR.

Abstract Background: A serum-based enzyme immunoassay (EIA) employing MAb-PAM4 demonstrates high specificity and sensitivity for detection of advanced pancreatic carcinoma (PC). We are currently exploring use of the immunoassay as a means to detect early-stage PC, as well as detection of nascent disease progression or relapse during routine post-treatment follow-up. We now also report initial results that provide correlation of serum PAM4-antigen levels with responses to radiolabeled PAM4 therapy. Methods: The PAM4-based EIA was employed for detection and quantitation of antigen in the serum of pancreatic carcinoma patients with known stage of disease (N=68). An additional 11 patients with stage-4 disease who participated in an ongoing phase-1b clinical trial to evaluate the combination of 90Y-hPAM4 IgG, (clivatuzumab tetrexate) radioimmunotherapy with low-dose, radiosensitizing, gemcitabine (Gem) were also examined. Serum specimens from this latter group were collected at baseline prior to treatment, at the end of the 4-week treatment cycle, and again at 4 weeks post-treatment (week 8 from baseline), when a CT scan was performed to determine tumor response. Results: Overall, the sensitivity of the immunoassay for detection of PC was 81%, with a 5% false-positive rate for the healthy volunteers (N=19). When examined by stage of disease, sensitivity rates were 91%, 86% and 62% for stages 3/4 advanced disease, stage-2, and stage-1 PC, respectively. For those patients who had treatment with 90Y-hPAM4 + Gem, a decrease in antigen levels greater than 40%, observed at the end of the treatment cycle (4 weeks), with sustained decline in the antigen levels at the 8-week post-treatment evaluation, provided presumptive evidence of tumor response, either stable disease (SD, N=2) or partial response (PR, N=5) by CT RECIST criteria. None of the patients with progressive disease (N=4) had a sustainable decrease in serum levels of PAM4-antigen, whereas all of the responders did. The median decrease in serum PAM4-mucin levels at the 8-week follow-up evaluation timepoint was 54% (range: 47% - 98% decrease), with a median tumor response (only SD and PR responders) being a decrease in tumor size of 37% (range: 46% decrease to 4% increase), from a baseline median primary tumor size of 10.0 cm (range 2.6 - 12.1 cm). Conclusions: These results suggest that the PAM4-based immunoassay should be further evaluated for use in the detection of early pancreatic carcinoma, as well as to evaluate tumor response to therapy. (Supported in part by grant CA096924 from the NIH.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4622.

Abstract Background: HLA-DR and CD74 are similarly, but not identically, expressed and induced by interferons on a variety of cells. Expression of both antigens on hematological malignancies led to their development as targets for antibody-based therapy. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab (Immunomedics Inc, Morris Plains, NJ), is in clinical evaluation for therapy of NHL, multiple myeloma (MM), and CLL after preclinical evidence of activity in these tumor types. A humanized anti-HLA-DR mAb, hL243γ4P (IMMU-114, Immunomedics) has demonstrated anti-tumor activity in vitro and in vivo, and clinical evaluation is planned. In addition to expression in hematologic cancers, these antigens are expressed on the surface of other tumor types, including melanoma and renal cell carcinoma, and in the cytoplasm of others, including pancreatic and colonic carcinomas, and glioblastomas (GBM). Methods: We examined whether the ability of anti-HLA-DR and anti-CD74 mAbs to kill cancer cells can be increased by using IFNγ as an inducer of antigen expression. Using a panel of diverse cancer cell lines (including NHL, MM, GBM, and pancreatic and colonic carcinomas), we examined IFNγ-induced changes in surface and cytoplasmic HLA-DR and CD74 expression. Sensitivity of malignant cells to milatuzumab and hL243γ4P was assessed with and without INFγ by cytotoxicity assays. Results: Without IFNγ surface expression of HLA-DR and CD74 were present on 2/2 NHL, 2/2 MM, and only weakly positive on 2/2 GBM cell lines. Surface CD74 and HLA-DR were weak or undetectable on 4/4 colon and 4/4 pancreatic carcinomas. Cytoplasmic CD74 and HLA-DR were seen in NHL, MM, GBM, and 1/4 colon and 1/4 pancreatic (CD74 only) carcinomas. Two-day incubation with IFNγ increased surface and cytoplasmic expression of both HLA-DR and CD74 in all the NHL and GBM, and 3/4 pancreatic cancer lines, but not MM cell lines. In all 4 colon lines, IFNγ increased cytoplasmic expression of both antigens, and surface expression of HLA-DR in 3/4 and CD74 in 2/4. Upregulation of HLA-DR and CD74 ranged from 23-3700%. Increased killing by both hL243γ4P (58%) and milatuzumab (33%) was seen in vitro after INFγ exposure in WSU-FSCCL NHL cells. No cell killing was observed using these mAbs in vitro on U118 (GBM), Capan-1 (pancreatic carcinoma), or LoVo (colon carcinoma), despite upregulation of the antigens in these cell lines. A CD74-transfected version of the U118 GBM cell line has been prepared for comparison of milatuzumab sensitivity based on antigen density only. Conclusions: Cell surface and cytoplasmic expression of CD74 and HLA-DR are increased on cell lines from a variety of cancer types after INFγ exposure. This increased expression correlates with increased toxicity of anti-HLA-DR and anti-CD74 mAbs in a NHL cell line, and is under evaluation in other cancer types. These studies could prove useful in predicting the potential benefit of combined INFγ and mAb therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5341.

4613 Background: Invasive pancreatic carcinoma is a virtually lethal disease, mostly because of the failure to detect it at a sufficiently early timepoint for successful treatment. Our laboratory has identified a unique biomarker detected by MAb PAM4 that shows high specificity for a mucin glycoprotein expressed by pancreatic carcinoma (PC). While identified in almost 90% of PC and its precursor lesions, the antigen is not detectable in normal pancreas. We are investigating this biomarker for the early detection of PC. Methods: Both immunohistochemical (IHC) and enzyme immunoassay (EIA) were employed for detection and/or quantitation of PAM4-mucin in tissue and sera, respectively. Results: We have extended our prior IHC results with precursor lesions (Clin Cancer Res 2007;13:7380–7); PAM4 gave an intense, diffuse labeling pattern in 81% of mucinous cystic neoplasms (MCN), with an additional 11% showing a focal pattern (n=27). Thus, a total of 92% of MCN showed evidence of PAM4-antigen expression. Of interest, a difference in the labeling pattern was observed in association with the grade of dysplasia, providing easy identification of MCN with high- grade dysplasia. We previously reported use of an EIA for quantitation of PAM4-antigen in sera. The assay demonstrated a sensitivity and specificity of 77% and 95%, respectively, for identification of PC (J Clin Oncol, 2006;24:252–8). We have now confirmed these results in a set of serum specimens (n= 49 PC, 13 normal) for which staging information was available. Overall specificity and sensitivity were 82% and 85%, respectively, calculated by ROC curve analysis (AUC=0.878±0.045; 95% CI=0.769–0.947). Although only a small number of specimens were from patients with stage I disease (n=12), 92% of these were above the cutoff value for positive response. A correlation was observed for average concentration of antigen in the circulation with stage of disease (R2=0.988). Conclusions: IHC and EIA results indicate that PAM4 identifies a biomarker for PC that is present at the earliest stages of neoplastic transformation, thus warranting controlled analyses of larger specimen numbers. (Supported in part by USPHS grant CA096924 from the NIH.) [Table: see text]

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable. By adding sphingomyelin to the culture medium, gemcitabine-induced cell death increased synergistically to >90%. Panc1 cells that survived high concentrations of gemcitabine had an increase in beta-galactosidase activity, a marker of senescence. The inclusion of sphingomyelin with gemcitabine reduced beta-galactosidase activity, as compared with cells treated with gemcitabine alone. Expression of p21(waf1/cip1) in both cell lines exposed to sphingomyelin, gemcitabine, and gemcitabine + sphingomyelin varied relative to the untreated group. C(8)-ceramide induced both cell death and senescence in a dose-dependent manner. These results indicate that gemcitabine induces senescence in pancreatic cancer cells and that sphingomyelin-enhanced chemosensitivity is achieved through reducing the induction of senescence by redirecting the cell to enter the apoptotic pathway. Ceramide levels seem to be critical to this decision, with cell cycle progression being uninhibited at low ceramide levels, senescence induced at moderate levels, and apoptosis initiated at high levels. Our results provide further evidence that targeting the sphingolipid metabolism is a means of enhancing the efficacy of chemotherapeutic agents.

Purpose: The anti-MUC1 monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (Pan IN) and intraductal papillary mucinous neoplasia (IPMN).Experimental Design: Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens were assessed by immunohistology for expression of the PAM4-reactive, non variable number of tandem repeats (VNTR), MUC1 epitope, and the VNTR epitope bound by the MA5 MAb.Results: The PAM4-reactive MUC1 epitope was not detected in normal pancreas but was expressed in 87% (48 of 55) of invasive pancreatic adenocarcinomas, including early stage 1 disease: PAM4 labeled 94% (44 of 47) of the earliest PanIN lesions, PanIN-1A and 1B, along with 91% (10 of 11) of PanIN-2, 40% (2 of 5) of PanIN-3, and 86% (31 of 36) of intraductal papillary mucinous neoplasia lesions. A mostly diffuse pattern of labeling was observed. A second, unrelated, anti-MUC1 MAb, MA5, showed considerably less sensitivity with early PanIN-1 lesions; only 61% (25 of 41) were positive and the labeling did not differentiate normal pancreas from PanINs.Conclusions: The results suggest that expression of the PAM4-reactive antigen may represent an early event in the development of invasive pancreatic adenocarcinoma, and is unrelated to the VNTR peptide core epitopes of MUC1. Detection of this biomarker using immunohistology, in vitro immunoassays, and in vivo antibody - based imaging may provide new opportunities for the early detection and improved diagnosis of pancreatic cancer.

4096 Background: Pancreatic cancer provides a major challenge in terms of diagnosis and treatment. We have developed an anti-MUC1 MAb, PAM4, which identifies an epitope that is more restricted to MUC1-expressed by pancreatic cancer than MUC1 from other forms of cancer. PAM4 has been studied for in vitro and in vivo detection and therapy of pancreatic cancer. Methods: The in vitro immunoassay consists of PAM4 as the capture reagent and an IgG fraction derived from a polyclonal, anti-MUC1 antiserum as the probe. For in vivo detection and therapy, PAM4 is either directly radiolabeled or used in a 2-step pretargeting protocol. Results: The PAM4-based immunoassay provided high sensitivity (77%) and specificity (95%), with a value ≥ 10.2 units/ml indicating a high likelihood of pancreatic cancer, as compared to normal and benign disease groups and non-pancreatic cancers. A direct pairwise comparison of the PAM4 and CA19–9 immunoassays for discrimination of pancreatic cancer and pancreatitis demonstrated a superior performance of the PAM4-immunoassay (P<0.003). Initial clinical studies with directly labeled 131I-PAM4 provided positive imaging in 8/10 patients, with one negative patient having only pancreatitis, and the other negative patient having a tumor that was MUC1-negative. A Phase I, dose-escalation study of 90Y-humanized PAM4 administered as a single dose to patients with advanced pancreatic cancer is in progress (Immunomedics, Inc), and has already achieved doses of 20 mCi/m2. Finally, pretargeting involving a bispecific MAb with one arm being PAM4 targeting MUC1 and the other arm capturing a hapten peptide carrying a radionuclide is under preclinical evaluation. This second generation targeting system has shown higher tumor/nontumor ratios and improved imaging (111In) as compared to directly radiolabeled PAM4. Conclusions: These results suggest that the PAM4-reactive MUC1 epitope may prove useful as a selective biomarker/target antigen for diagnosis, detection, imaging, and therapy of pancreatic cancer. (Supported in part by grants CA96924and CA98488 from the NIH). [Table: see text]

Ceramide is a bioactive lipid involved in the induction of apoptosis and is the precursor to several sphingolipids, including sphingomyelin, the gangliosides, and sphingosine. Ceramide production is increased in response to stress and toxic agents. Because modulation of ceramide levels has been shown to affect sensitivity and/or resistance to therapeutic agents, it will be important to assess the activity of sphingolipid metabolic pathways when investigating the mode of action of antitumor drugs. This chapter summarizes protocols for quantitating the level of apoptosis, the activities of acidic sphingomyelinase, neutral sphingomyelinase, glycosylceramide synthase, sphingomyelin synthase, and ceramidase, and the amount of ceramide in tumor xenografts in nude mice.

Several reports have appeared on the use of combined radioimmunotherapy (RAIT) and chemotherapy. The choice of drug to use with RAIT and how to space the two treatments has not been completely addressed. Because every patient's cancer presents with a specific molecular phenotype, we hypothesized that it may be necessary to tailor therapy based on specific gene expression. We addressed how the form of expression of a single gene, the p53 tumor suppressor, would impact the choice of agents, as well as sequence and spacing of agents. p53 regulates cell cycle arrest to allow for DNA repair after therapy‐induced small DNA damage or induction of apoptosis if damage is great and has been shown to affect chemo‐ and radiosensitivity of cancer cells. We established 3 stable p53 transfectants of the SKOV‐3 p53null parental line (p53wt, p53143mut or p53273mut). p53 expression was confirmed using flow cytometry, using the DO1 pan‐p53 Ab and the PAb240 anti‐p53mut Ab. The colorimetric MTT assay was then used to measure dose‐dependent growth inhibition from single modality chemotherapy (doxorubicin, carboplatin, paclitaxel or topotecan) or radioimmunotherapy (90Y‐RS‐7 IgG anti‐EGP1). The % survival vs. log [drug] were plotted to obtain the IC50. We then used a matrix design in which we varied the sequence of the first and second modality of treatment and the spacing between the 2 treatments to determine the most synergistic and antagonistic combinations for the parental SKOV‐3 and each of the 3 transfectants. The IC50 for each therapeutic agent varied as a function of the form of p53 expressed. For example, of the 4 lines, the p53wt transfectant was the most resistant to topotecan and the 143mut was the most resistant to carboplatin. The 273mut was quite sensitive to both doxorubicin and paclitaxel, whereas the p53null and wt were not. For multimodal treatments, most combinations of RAIT and chemotherapy resulted in a 30–40% growth inhibition (GI) and were either additive or moderately antagonistic. The 3 best (>60% GI) and 3 worst (<25% GI) combinations were identified and were unique to the parental p53null and to the 3 transfectants. Certain combinations showed clear synergy and others were antagonistic, with the first treatment modality blocking the growth inhibitory effects of the second treatment modality. The form of p53 expressed affects chemosensitivity and radiosensitivity and will influence optimal multimodal therapy with RAIT and chemotherapy and the dose‐schedule (sequential with RAIT first or with drug first) when more than 1 agent is used. © 2003 Wiley‐Liss, Inc.

PURPOSE:Monoclonal antibody PAM4 is reactive with the MUC1 mucin as expressed by >85% of human pancreatic cancers. Significant antitumor effects have been demonstrated using radiolabeled PAM4 for radioimmunotherapy (RAIT) of experimental pancreatic cancer. The goal of the present study was to determine whether the addition of low-dose (90)Y-PAM4 RAIT to a clinically relevant regimen of gemcitabine chemotherapy would provide enhanced antitumor efficacy over that observed by chemotherapy alone without the addition of significant toxicity to normal tissues.EXPERIMENTAL DESIGN:Mice bearing human pancreatic tumor xenografts (CaPan1) were administered three cycles of gemcitabine chemotherapy (1000 mg/m(2)/week for 3 weeks with 1 week off) concomitant with (90)Y-labeled PAM4 RAIT (25 micro Ci; 10% of the single agent MTD) given at weeks 0, 4, and 7. Control groups of mice received chemotherapy alone, (90)Y-PAM4 RAIT alone, or an equidose of (90)Y-labeled nontargeting control antibody with and without gemcitabine.RESULTS:Mice that received (90)Y-PAM4 RAIT with gemcitabine had tumors that were significantly smaller in size than all of the other treatment groups (P < 0.005). A median survival of 24 weeks was achieved in mice that received the combined treatment versus 10 weeks for mice that received only gemcitabine (P < 0.001) and 16 weeks for mice that received only (90)Y-PAM4 RAIT (P < 0.040). The combined treatment regimen was well tolerated.CONCLUSIONS:A combined chemoimmunotherapy and RAIT approach using gemcitabine and low-dose (90)Y-PAM4 provided significantly increased antitumor efficacy than was observed for each treatment arm given alone. Importantly, the enhanced antitumor efficacy was achieved with minimal toxicity to normal tissues. These studies provide justification for clinical trials using the combined modality treatment for patients with pancreatic cancer.

We examined the genetic and biochemical bases for drug resistance and the order of appearance of different mechanisms underlying the increasingly more resistant murine erythroleukemia cell lines established in Adriamycin (ADR). In the first-step low-level resistant cell line PC4-A5 (able to grow in 5 ng/mL ADR), there was a 2-fold reduction in topoisomerase IIalpha and topoisomerase IIbeta mRNA levels, as well as topoisomerase IIalpha protein and activity levels as compared with the parental cell line. The topoisomerase IIalpha activity levels remained reduced as the cells became increasingly more resistant. In contrast, the topoisomerase II mRNA and protein levels returned to approximately the parental levels in resistant cells growing in higher drug concentrations (40-160 ng/mL). Parental cells expressed the multidrug resistance protein (MRP), but beginning with PC4-A5 MRP expression decreased and remained reduced in increasingly resistant cell lines. At high levels of ADR resistance, the cells expressed the mdr3 gene concomitant with the appearance of vincristine resistance and energy-dependent daunomycin and vincristine efflux. Glutathione levels, internal pH, and expression of the major vault protein (MVP) remained unchanged in all cell lines. Fluorescence microscopy revealed no alterations in daunomycin distribution or vesicle numbers between the parental and resistant cell lines. Different resistance mechanisms emerge sequentially as cells become more resistant to ADR; the mechanisms are retained during the development of multidrug resistance (MDR). In intermediate-level MDR cell lines (PC4-A10 and PC4-A20), resistance involves an as yet undetermined mechanism(s).

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)