Characterization of lymphocyte transformation induced by zinc ions.

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn(++). Examination of the effects of other divalent cations reveals that 0.01 mM Hg(++) also stimulates lymphocyte DNA synthesis. Ca(++) and Mg(++) do not affect DNA synthesis in this culture system, while Mn(++), Co(++), Cd(++), Cu(++), and Ni(++) at concentrations of 10(-7)-10(-3) M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2-3 days with Zn(++) and these processes reach a maximum rate after 6 days. The increase in Zn(++)-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn(++)-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn(++) must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn(++) inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.

Sexual dimorphism in developmental and diet-dependent circulating retinol binding protein 4.

OBJECTIVE: Retinol binding protein 4 (RBP4) transports vitamin A (Retinol) in the blood and contributes mechanistically to the linkage between obesity, insulin resistance and associated comorbidities including type 2 diabetes mellitus, coronary artery and neoplastic diseases. Circulating RBP4 levels have variably been associated with body mass and gender differences. Many of these differences have been demonstrated after limited dietary interventions, and/or at single unique time points. This study investigated the impact of sex and age as biologic variables as well as high versus low fat diets on development of obesity, RBP4 levels and insulin resistance in C57BL/6J mice. METHODS: Male and female C57BL/6J mice were fed for 400 days with either low or high fat diets. Female mice were also evaluated on same diets after ovariectomy or sham ovariectomy. Mice were monitored for changes in weight, circulating levels RBP4, glucose and insulin at 100-day intervals and also by 2-hour glucose tolerance tests. RESULTS: All mice on low or high fat diets gained weight. Mice on high fat diets showed significantly greater weight gain than those on low fat. Male mice showed significantly greater weight gain compared with females on corresponding diet. Male mice compared with females already showed significantly higher RBP4 levels even before starting diets. Sex differences were maintained for more than 1 year. Gender differences in RBP4 were associated with significant differences in development of glucose intolerance and insulin resistance. CONCLUSIONS: Male compared with female C57BL/6J mice show significant gender differences in circulating RBP4 levels from 6 weeks of age, extending more than 1 year. Gender differences in RBP4 may be mechanistically associated with protection against glucose intolerance and insulin resistance. Targeting RBP4 pathways could be useful to disrupt gender differences in insulin resistance and disparities in comorbidities.

O6 alkylguanine-DNA alkyltransferase activity in human myeloid cells.

The association between alkylating agent exposure and acute nonlymphocytic leukemia in humans indicates that myeloid cells may be particularly susceptible to mutagenic damage. Alkylating agent mutagenesis is frequently mediated through formation and persistence of a particular DNA base adduct, O6alkylguanine, which preferentially mispairs with thymine rather than cytosine, leading to point mutations. O6alkylguanine is repaired by O6alkylguanine-DNA alkyltransferase (alkyltransferase), a protein that removes the adduct, leaving an intact guanine base in DNA. We measured alkyltransferase activity in myeloid precursors and compared it with levels in other cells and tissues. In peripheral blood granulocytes, monocytes, T lymphocytes, and B lymphocytes, there was an eightfold range of activity between individuals but only a twofold range in the mean activity between cell types. Normal donors maintained stable levels of alkyltransferase activity over time. In bone marrow T lymphocytes and myeloid precursors, there was an eightfold range of alkyltransferase activity between donors. Alkyltransferase activity in the two cell types was closely correlated in individual donors, r = 0.69, P less than 0.005, but was significantly higher in the T lymphocytes than the myeloid precursors, P less than 0.05. Liver contained the highest levels of alkyltransferase of all tissues tested. By comparison, small intestine contained 34%, colon 14%, T lymphocytes 11%, brain 11%, and myeloid precursors 6.6% of the activity found in liver. Thus, human myeloid precursors have low levels of O6alkylguanine-DNA alkyltransferase compared with other tissues. Low levels of this DNA repair protein may increase the susceptibility of myeloid precursors to malignant transformation after exposure to certain alkylating agents.

Association of poly(adenosine diphosphoribose) synthesis with DNA damage and repair in normal human lymphocytes.

A permeable cell technique was used to measure the alterations in synthesis of DNA and poly-(adenosine diphosphoribose) in normal human lymphocytes after treatment of the cells with different types of DNA-damaging agents. The lymphocytes showed an abrupt increase in the unscheduled synthesis of DNA and poly(adenosine diphosphoribose) in response to ultraviolet (UV) irradiation. The increases were apparent within 1 h and reached a maximum between 2 and 4 h after irradiation. The magnitude of the increases in DNA and poly(adenosine diphosphoribose) synthesis was dependent upon the UV dose. Alkaline CsCl gradient studies, with bromodeoxyuridine triphosphate density labeling of DNA, demonstrated that the unscheduled DNA synthesis, which occurred in response to UV irradiation, was actually a result of the repair mode of DNA synthesis. Similar increases in DNA synthesis, and poly(adenosine diphosphoribose) synthesis occurred when lymphocytes were treated with several other DNA-damaging agents, including bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine or N-acetoxyacetyl aminofluorene. Treatment of lymphocytes with DNase, under conditions which allowed degradation of cellular DNA, also resulted in increased synthesis of poly(adenosine diphosphoribose). Cycloheximide did not inhibit the increase in synthesis of DNA or poly(adenosine diphosphoribose) that occurred in response to treatment with the DNA-damaging agents.

Role of nicotinamide adenine dinucleotide and adenosine triphosphate in glucocorticoid-induced cytotoxicity in susceptible lymphoid cells.

The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.

Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

Description of a permeable eukaryotic cell system to study agents affecting DNA synthesis: demonstration that cytembena is a direct inhibitor of replicative DNA synthesis.

Cells were made permeable to DNTP's by a 15-minute treatment with 0.01 M Tris/HCl (pH 7.8), 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol, and 4 mM MgCl2 at 4 degrees C. These cells used exogenously supplied dNTP's to carry out semiconservative, replicative DNA synthesis. This system could be used to identify inhibitors of DNA synthesis and to determine whether chemotherapeutic agents affect precursor synthesis or whether they have a direct effect on the DNA replication complex. Thus DNA synthesis in the permeable cells was not inhibited by hydroxyurea or by the nucleoside analogue arabinofuranosyl cytosine. In contrast, the active form of the nucleoside analogue, arabinofuranosyl cytosine triphosphate, and daunorubicin promptly inhibited DNA synthesis in the permeable cells. Cytembena, sodium cis-beta-4-methoxybenzoyl-beta-bromoacrylate, also inhibited DNA synthesis in the permeable cells; this demonstrated that this agent functioned as a direct inhibitor of the DNA replication complex.

Growth characteristics of MOPC-315 plasmacytoma and response to anticancer agents.

We examined the growth characteristics and response to anticancer agents of the murine plasmacytoma MOPC-315, a rapidly growing and widely disseminating tumor syngeneic in BALB/c mice. The doubling time for the tumor was approximately 24 hours, and about 1 in 100 cells was tumorigenic. We developed a spleen colony assay for the clonogenic component of the tumor and used it to define the dose-response relationships for various anticancer agents, including melphalan, cyclophosphamide, 1,3-bis(chloroethyl)-1-nitrosourea, 5-fluouracil, gamma-radiation, methotrexate, methylprednisolone, cytosine arabinoside, and vincristine.

Factors affecting topotecan-induced programmed cell death: adhesion protects cells from apoptosis and impairs cleavage of poly(ADP-ribose)polymerase.

We have evaluated the influence of anchorage status together with endogenous levels of bcl-2 family members on the ability of the topoisomerase I inhibitor, topotecan (TPT), to induce programmed cell death (PCD) in human colon, breast, lymphoid, and cervical cancer cell lines. As part of this study, we assessed the use of measuring poly(ADP-ribose) polymerase (PARP) cleavage by Western blot, as an index of apoptosis, relative to measuring chromatin condensation by acridine orange analysis. Our results show a strong correlation between both assays, indicating that PARP cleavage is an accurate method to examine PCD. We have encountered a strong association between cell attachment and sensitivity to TPT-induced PCD. Cells growing attached to flasks appear to be relatively more resistant than suspension-growing cells in spite of endogenous bcl-2, bax, or bcl-x levels. Furthermore, we demonstrate that interference with attachment status alters the sensitivity of cells to TPT-induced PCD. Although cell attachment to ProNectin F confers protection against TPT-induced chromatin condensation and cleavage of PARP, cell detachment by poly(2-hydroxyethyl methacrylate) stimulates TPT-induced PCD and PARP cleavage.

Synergistic antileukemic effect of 6-aminonicotinamide and 1,3-bis(2-chloroethyl)-1-nitrosourea on L1210 cells in vitro and in vivo.

A series of nicotinamide analogs were evaluated for their ability to inhibit L1210 cell poly(adenosine diphosphoribose) polymerase, and also for their ability to potentiate the cytocidal effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), nitrogen mustard, and gamma-irradiation. In L1210 cells growing in culture and in vivo. In vitro, nicotinamide, 5-methylnicotinamide, 6-aminonicotinamide, and benzamide effectively inhibited L1210 cell poly(adenosine diphosphoribose) polymerase; 1-methylnicotinamide, nicotinic acid, and benzoic acid did not. In culture, 6-aminonicotinamide potentiated the cytocidal effect of BCNU; however, it did not significantly potentiate the effects of nitrogen mustard or gamma-irradiation in vivo, both 6-aminonicotinamide and nicotinamide potentiated the cytocidal effect of BCNU; however, the concentrations of nicotinamide required for this effect were 10- to 20-fold higher than those of 6-aminonicotinamide. None of the analogs significantly potentiated the in vivo effect of nitrogen mustard or gamma-irradiation. Treatment of L1210-bearing mice with varying combinations of BCNU and 6-aminonicotinamide produced a synergistic increase in life span; in some cases, the combination led to the production of long term disease-free survivors.

Inhibition of etoposide (VP-16)-induced DNA recombination and mutant frequency by Bcl-2 protein overexpression.

Bcl-2 has been shown to inhibit apoptosis induced by several anticancer agents and to cause a dissociation between etoposide (VP-16)-induced protein-cross-linked DNA strand breaks and VP-16-induced cell death. We suggested previously that VP-16-induced cytotoxicity is mediated by a series of events leading from cleavable complex formation to aberrant DNA recombination, as measured by sister chromatid exchange (SCE) and Southern blot analysis of the hypoxanthine phosphoribosyl transferase (hprt) gene mutations. To further evaluate this hypothesis and to determine whether Bcl-2 could affect any steps leading to the aberrant DNA recombination process, we stably transfected an expression vector containing human Bcl-2 cDNA into V79 Chinese hamster cells. This transfection resulted in overexpression of the Bcl-2 gene product. We subsequently quantitated the relationship between VP-16-induced cytotoxicity, DNA strand breaks, SCE, and mutant frequency at the hprt locus in these Bcl-2-overexpressing cells. Two independent Bcl-2-overexpressing cell lines, BCL2/2 and BCL2/4, showed 3-5 times higher survival at 15 microM VP-16 compared with parental V79 cells or control NeoR cells that were obtained by transfecting V79 cells with the expression vector containing the G-418 resistance gene only. DNA single-strand breaks induced by VP-16 were similar in parental V79, control NeoR, BCL2/2, and BCL2/4 cells. In contrast, VP-16 induced significantly less SCE in Bcl-2-overexpressing cell lines compared with parental V79 and control NeoR cells. The SCE/chromosome induced by 15 microM VP-16 were 0.65, 0.42, 0.09, and 0.10, respectively, in V79, NeoR, BCL2/2, and BCL2/4. In addition, there was an excellent correlation between VP-16-induced SCE and cytotoxicity in all cell lines. Furthermore, VP-16-induced mutant frequencies at the hprt locus were 5-10 times less in BCL-2/2 and BCL-2/4 cells than those observed in the V79 or NeoR control cells. These results indicate that overexpression of Bcl-2 is associated with reduction in VP-16-induced genetic recombination, mutation, and cytotoxicity. Moreover, they suggest that Bcl-2 modulates cytotoxicity of VP-16 between cleavable complex formation and subsequent induction of DNA recombination events. Thus, our results provide important support for the hypothesis that VP-16-induced cytotoxicity is associated with aberrant recombination events, including gene deletions and rearrangements.

Induction of M(r) 78,000 glucose-regulated stress protein in poly(adenosine diphosphate-ribose) polymerase- and nicotinamide adenine dinucleotide-deficient V79 cell lines and its relation to resistance to the topoisomerase II inhibitor etoposide.

Cell lines deficient in poly(ADP-ribose) synthesis due to enzyme deficiency (ADPRT54 and ADPRT351) or substrate deficiency (N2, N3, and N4) are resistant to topoisomerase II-directed agents, including etoposide (VP-16), N-[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 NAD levels return to control levels during growth in nicotinamide-containing complete medium. Poly(ADP-ribose) polymerase-deficient cell lines show constitutively increased levels of a protein at M(r) 78,000 on Coomassie blue-stained, sodium dodecyl sulfate-polyacrylamide gels that was subsequently confirmed with monoclonal antibodies to be M(r) 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 association between deficiency of the NAD-poly(ADP-ribose) synthesis system, induction of GRP78 synthesis, and resistance to VP-16.

Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines.

Mutant Chinese hamster V79 cells selected for alterations in poly(ADP-ribose) metabolism were shown to be resistant to epipodophyllotoxin (VP-16)-induced cytotoxicity. Cell lines ADPRT 54 and ADPRT 351 have reduced activity of poly(ADP-ribose) polymerase. N2, N3, and N4 cell lines grow in the absence of nicotinamide, with total NAD levels 1.5-3% of those found in parental V79 cells grown in complete medium. When grown in complete medium, the mutant cell lines are 2.3- to 9.6-fold resistant to VP-16-induced cytotoxicity. All of the cell lines respond to VP-16 treatment by formation of protein-cross-linked DNA strand breaks. Upon drug removal, all the cell lines reverse the DNA strand breaks at similar rates. Our studies show a clear dissociation between induction of DNA strand breaks and cytotoxicity. However, there is a good correlation between drug-induced sister chromatid exchanges and cytotoxicity. Thus, N3 cells, with low levels of VP-16-induced sister chromatid exchanges, show reduced levels of cytotoxicity relative to parental V79 cells, despite the fact that both cell lines show similar levels of VP-16-induced protein-cross-linked DNA strand breaks. Additional studies show that the time course of VP-16-induced cytotoxicity correlated better with the time course of sister chromatid exchange formation than with protein-cross-linked DNA strand break formation. These studies provide strong support for the proposal that VP-16-induced cytotoxicity involves the induction of sister chromatid exchanges. Thus, we suggest that drug-induced stabilization of topoisomerase II-DNA complexes stimulates induction of sister chromatid exchanges, which consequently lead to cell death.

Topoisomerase II-dependent and -independent mechanisms of etoposide resistance in Chinese hamster cell lines.

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 contributed 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 drug-mediated 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 resistance to drug-mediated DNA scission, and a second mechanism independent of the effects of the drug on topoisomerase II.

Sensitive enzymatic cycling assay for glutathione: measurements of glutathione content and its modulation by buthionine sulfoximine in vivo and in vitro in human colon cancer.

We have measured glutathione content in small tissue samples derived from biopsies of primary and metastatic human colon tumors and from colon cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10(-14) mol) from perchlorate extracts of tissue samples weighing less than 1 mg wet weight. Glutathione was stable in these acid extracts for at least 6 months when stored at -80 degrees C. A survey of normal tissues in mice, rats, and some human tissues showed considerable variation in GSH content of different tissues but generally similar levels were identifiable for the same tissues from different species. The highest GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1.8 nmol/mg protein in rat skeletal muscle. High GSH levels were also determined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies. These studies revealed a marked inter-individual difference in tumor GSH content, as well as a difference in GSH content between tumor deposits at different metastatic sites in the same individual. These results indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase, was shown to produce almost complete depletion of GSH in four different human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in human colon cancer grown as xenografts in athymic animals.

Topotecan increases topoisomerase IIalpha levels and sensitivity to treatment with etoposide in schedule-dependent process.

To elucidate the effect of topoisomerase (Topo) I inhibitors in the modulation of Topo II levels and sensitivity to Topo II-directed drugs, athymic mice bearing SW480 human colon cancer xenografts were treated with simultaneous, subsequent, or distant doses of topotecan and etoposide. This in vivo study demonstrates that simultaneous administration of topotecan and etoposide results in an antagonistic response. In contrast, inhibition of Topo I by topotecan results in a compensatory increase in Topo II alpha levels associated with increasing sensitivity of tumors to subsequent treatment with the Topo II inhibitor etoposide. Furthermore, we show that Topo II alpha levels decline 5 days after the last dose of topotecan, resulting in restoration of the original response of the xenografts to etoposide. Thus, this study emphasizes the critical role of schedule dependency to optimize the effectiveness of combination chemotherapy with Topo I and Topo II inhibitors.

Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment.

Apoptosis induced by numerous cancer chemotherapeutic and other toxic agents has been shown to proceed through a cascade of proteases, now termed caspases, culminating in cleavage of a set of proteins. The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular caspases has been assessed during the rapid apoptosis in murine lymphoma L5178Y-R cells. Cells were exposed to combinations of Pc 4 and activating red light that result in > or =90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 microM Pc 4 and either 2.1 or 3 kJ/m2, which kill 90 or 99.9% of the cells, oligonucleosomal fragmentation was visible on agarose gels as early as 60 or 30 min after PDT, respectively. To assess caspase activation, cells were harvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavage of the normally Mr 116,000 PARP into fragments of Mr approximately 90,000 was observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the Mr approximately 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) Mr 100,000 protein, tentatively identified as topoisomerase I, were maintained in cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable, irreversible caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, whereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3 and/or other similar caspases.

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.