Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase.

BACKGROUND: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. METHODS: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. RESULTS: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS-), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS- patients receiving at least four doses at 320 IU m(-2) was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. CONCLUSION: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

Analysis of DNA attached to the chromosome scaffold.

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.

Inhibition of mTOR restores cisplatin sensitivity through down-regulation of growth and anti-apoptotic proteins.

We show that cisplatin resistance in certain lung cancer cell lines can be reversed through inhibition of mTOR (mammalian Target of Rapamycin). These cell lines appear to possess high levels of phospho-mTOR, phospho-AKT and other growth-related proteins, such as hTERT (human telomerase reverse transcriptase), and Cyclin D3 which decrease upon inhibition of mTOR. Interestingly in one cisplatin resistant cell line which expresses BCL2/BCLxL, treatment with mTOR inhibitor (CCI-779) results in decreased levels of these anti-apoptotic proteins and may contribute to increasing apoptosis. Moreover, continuous exposure to CCI-779 was found to increase the expression of the multi-drug resistant P-gp1(P-gycoprotein1) efflux pump and therefore should be taken into consideration when designing clinical trials with this compound.

MRP class of human ATP binding cassette (ABC) transporters: historical background and new research directions.

1. The adenosine triphosphate (ATP) binding cassette (ABC) transporters form one of the largest protein families encoded in the human genome, and more than 48 genes encoding human ABC transporters have been identified and sequenced. It has been reported that mutations of ABC protein genes are causative in several genetic disorders in humans. 2. Many human ABC transporters are involved in membrane transport of drugs, xenobiotics, endogenous substances or ions, thereby exhibiting a wide spectrum of biological functions. According to the new nomenclature of human ABC transporter genes, the 'ABCC' gene sub-family comprises three classes involving multidrug resistance-associated proteins (MRPs), sulfonylurea receptors (SURs), and a cystic fibrosis transmembrane conductance regulator (CFTR). 3. Molecular cloning studies have identified a total of ten members of the human MRP class including ABCC11, ABCC12, and ABCC13 (pseudo-gene) that have recently been characterized. 4. This review addresses the historical background and discovery of the ATP-driven xenobiotic export pumps (GS-X pumps) encoded by MRP genes, biological functions of ABC transporters belonging to the MRP class, and regulation of gene expression of MRPs by oxidative stress.

Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

Overexpression of the multidrug resistance gene mdr3 in spontaneous and chemically induced mouse hepatocellular carcinomas.

Overexpression of a family of plasma membrane glycoproteins, known as P-glycoproteins, is commonly associated with multidrug resistance in animal cells. In rodents, three multidrug resistance (mdr or pgp) genes have been identified, but only two can confer the multidrug resistance phenotype upon transfection into animal cells. Using the RNase protection method, we demonstrated that the levels of three mdr gene transcripts differ among mouse tissues, confirming a previous report that the expression of these genes is tissue specific (J.M. Croop, M. Raymond, D. Huber, A. DeVault, R. J. Arceci, P. Gros, and D. E. Housman, Mol. Cell. Biol. 9:1346-1350, 1989). The levels of mdr transcripts were determined for mouse liver tumors spontaneously arising in both C3H/HeN and transgenic animals containing the hepatitis B virus envelope gene and for tumors induced by two different carcinogenic regimens in C57BL/6N and B6C3-F1 mice. The mdr3 gene was overexpressed in all 22 tumors tested. Our results demonstrate that overexpression of the mdr3 gene in mouse liver tumors does not require exposure of the animals to carcinogenic agents and suggest that its overexpression is associated with a general pathway of hepatic tumor development. The overexpression of the mdr3 gene, which is the homolog of human mdr1 gene, in hepatocellular carcinomas may be responsible for the poor response of these tumors to cancer chemotherapeutic agents.

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.

Relationship of Metabolic Alterations and PD-L1 Expression in Cisplatin Resistant Lung Cancer.

Despite numerous reports on immune checkpoint inhibitor for the treatment of non-small cell lung cancer (NSCLC), the response rate remains low but durable. Thus cisplatin still plays a major role in the treatment of NSCLC. While there are many mechanisms involved in cisplatin resistance, alteration in metabolic phenotypes with elevated levels of reactive oxygen species (ROS) are found in several cisplatin resistant tumors. These resistant cells become more reliant on mitochondria oxidative metabolism instead of glucose. Consequently, high ROS and metabolic alteration contributed to epithelial-mesenchymal transition (EMT). Importantly, recent findings indicated that EMT has a crucial role in upregulating PD-L1 expression in cancer cells. Thus, it is very likely that cisplatin resistance will lead to high expression of PD-L1/PD-1 which makes them vulnerable to anti PD-1 or anti PD-L1 antibody treatment. An understanding of the interactions between cancer cells metabolic reprogramming and immune checkpoints is critical for combining metabolism targeted therapies with immunotherapies.

Expression of the mdr (P-glycoprotein) gene in Chinese hamster digestive tracts.

P-glycoprotein has been shown to be responsible for multidrug resistance in mammalian cells. However, its physiological roles in normal cells are not known. The gene encoding this protein has been shown to express at a relatively high level in human digestive tracts. In the present study, in situ hybridizations were employed to determine the expression of this gene in gastrointestinal tissues. Epithelial cells in the villi of small intestine, colon, and stomach were rich in the P-glycoprotein gene transcript. Observations were consistent with the idea that the P-glycoprotein plays a role in detoxification by pumping potentially harmful compounds into the lumen of digestive tracts in animals.

Altered gel electrophoretic mobility of bleomycin-mediated release of nucleosomal DNA.

Nucleosomal DNA isolated from bleomycin-treated nuclei shows retarded electrophoretic mobility in neutral gels as compared with the sample isolated from micrococcal (or endogenous) nuclease-digested nuclei. The retardation in electrophoretic mobility is probably due to the presence of single-strand regions in the DNA duplex of bleomycin-treated samples as determined by single-strand-specific S1 nuclease digestion and buoyant density measurements. This observation argues against the possibility that the generation of nucleosomal DNA following drug treatment of nuclei is due to an activation of endogenous nuclease by bleomycin and strongly suggests that the drug has a unique feature of action on chromatin.

Preferential damage of active chromatin by bleomycin.

Preferential sensitivity of specific genes in chromatin to the action of bleomycin in different cell types was investigated using restriction enzyme digestion of DNA from drug-treated nuclei in combination with the Southern blotting procedure. The results show that the highly active ovalbumin gene in laying hen oviduct nuclei is more sensitive than the inactive globin gene to the digestion by bleomycin. Conversely, in red blood cells, bleomycin preferentially destroys the globin gene but not the ovalbumin gene. Therefore, bleomycin, like nucleases, can preferentially destroy DNA sequences in chromatin with open configuration, although the mode of action of this drug on DNA is drastically different from those of known nucleases.

Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification.

Double minutes (DM) have been associated with gene amplification in drug-resistant cells and tumor cells. However, the mechanisms by which DM are formed have not been elucidated. We present here a model to describe a possible mechanism of DM formation based on the observations made in two independent early drug-selected multidrug-resistant cell lines and from in vitro somatic cell fusion experiments between synchronized S- and M-phase cells. The multidrug-resistant cell lines contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic analyses of cells at early stages of selection revealed the presence of a number of micronuclei in a subpopulation of these cells. These micronuclei were often asynchronous in their progression through the cell cycle. As a result, premature condensation of micronuclear chromatin was often observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes displays a striking resemblance to DM structures. These DM-like structures are linked by replicating DNA as revealed by DNA labeling experiments. Somatic cell hybrids between S- and M-phase cells when grown in vitro demonstrated that S-phase prematurely condensed chromatin indeed gives rise to extra chromosomal structures in the successive cell generations. It is hypothesized that distinct DM-like structures may arise from the partially replicated and prematurely condensed S-phase chromosomes following their liberation as extra chromosomal entities after replication and/or recombination in the succeeding division cycle(s). The enrichment for DM containing specific genes in drug-resistant cells may result from the subsequent drug selections.

Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea in human glioma cells.

Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), an alkylating antitumor agent the primary target of which has been thought to be DNA, resulted in elevated expression of mRNA for multidrug resistance-associated protein (MRP) within the first 2 h and then a decrease in expression 24 h after the treatment. Western blot analyses revealed that levels of MRP in these ACNU-treated cells paralleled mRNA levels. Membrane vesicles prepared from ACNU-treated cells also displayed elevated transport activities for leukotriene C4, a known substrate for MRP. Gamma-glutamylcysteine synthetase (gamma-GCS) mRNA expression was coinduced with MRP by ACNU. Because gamma-GCS is the rate-limiting enzyme involved in the de novo biosynthesis of glutathione, increases in glutathione were also transiently induced by ACNU. These results demonstrate for the first time that the expression of functional MRP and gamma-GCS can be transiently coinduced by ACNU. Multiple short exposures (1 h) of ACNU following a long duration (1 week) of drug-free conditions resulted in the development of an ACNU-resistant population (designated A172R) that overexpressed MRP/gamma-GCS mRNA and had elevated transport activities for leukotriene C4. A172R exhibited cross-resistance to the antitumor drug doxorubicin and heavy metal sodium arsenate but not to cisplatin. Our results also demonstrate that intermittent treatments of human glioma cells with ACNU can lead to the development of MRP-related multidrug resistance. These results, taken together, reveal a possible new mechanism of the development of drug resistance for the antitumor nitrosoureas.

Effect of bleomycin on the synthesis and function of RNA.

Bleomycin inhibits cellular RNA synthesis and the inhibition is nonspecific. The ratio of polyadenylate- [poly(A)] containing RNA to non-poly(A)-containing RNA in the drug-treated human lymphocytic cells, line Wil2, was the same as that in untreated cells. Poly(A) RNA isolated from untreated cells was used as a template for reverse transcriptase to synthesize complementary DNA, which was then used as a probe to assay the sequence diversity of poly(A)RNA's from treated and untreated cells. It was found that essentially all of the poly(A) RNA's in the untreated cells were also present in the treated cells. The effect of bleomycin on the biological activity of messenger RNA (mRNA) was tested with globin mRNA in a wheat germ embryo translation system. Although bleomycin inhibited protein synthesis at high concentrations, the inhibition was not due to a modification of mRNA. This was evidenced by the fact that no decrease in the ability of mRNA to function in the test system was found when globin mRNA was pretreated with high concentrations of bleomycin followed by removal of the drug.

Frequent coordinated overexpression of the MRP/GS-X pump and gamma-glutamylcysteine synthetase genes in human colorectal cancers.

We have recently shown that multidrug resistance-associated protein (MRP) and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit genes are coordinately overexpressed in cisplatin-resistant human leukemia cells (T. Ishikawa et al. J. Biol. Chem., 271: 14981-14988, 1996). Using the RNase protection assay, we examined expression levels of these genes in colon tumor and nontumorous biopsy specimens from 32 cancer patients who had not been treated with chemotherapy. Increased mRNA levels (P < 0.001) of MRP and gamma-GCS genes were observed in 16 (50%) and 20 (62%) tumor samples, respectively. More importantly, all of the 16 (100%) MRP-overexpressing tumor specimens also exhibited higher levels of gamma-GCS mRNA than those in the matched nontumorous specimens. The correlation coefficient between MRP and gamma-GCS mRNA levels was r = 0.78 for all of the tumor samples studied. These results strongly suggest that MRP and gamma-GCS genes are coordinately up-regulated during colorectal carcinogenesis.

Methylation of the alpha-fetoprotein gene in productive and nonproductive rat hepatocellular carcinomas.

The extent of methylation of Hpall-Mspl and Hhal sites in DNAs isolated from normal rat livers and from the transplantable hepatocellular carcinomas (THC) THC 7777 and THC 252 was compared. It was found that the overall level of methylation of the internal cytosine in CCGG sequences was lower in the THC DNAs than in the normal liver DNAs. This difference could also be detected in the extent of methylation of CCGG sites flanking a 400-base pair repetitive sequence. Examination of methylation of specific sites within the alpha-fetoprotein gene revealed differences between the DNAs that appear to reflect both the level of activity of the gene and the overall level of methylation of cellular DNA. This gene, which is repressed in normal adult liver and the nonproductive THC 252 and highly active in the productive THC 7777 (S. Sell et al., Cell Biol. Int. Rep., 4: 235-254, 1980), contains several CCGG sites that are methylated in both normal liver and THC 252 DNA but not in THC 7777 DNA. However, Hhal (GCGC) sites in the alpha-fetoprotein gene were less methylated in both hepatoma DNAs than in liver DNA, which the exception of one site in the productive tumor found to be no longer methylated.

Expression of endogenous murine leukemia virus in transplantable tumor cells and DNA methylation of the viral sequences.

A cell line derived from early passage of a transplantable tumor in athymic mice that initially had been injected with human breast adenocarcinoma cells produced a substantial amount of RNA sequences related to the Kirsten murine leukemia virus. The cell line derived from later passage of transplantation of these tumors diminished viral RNA production. Expression of the viral RNA sequences in these cell lines is inversely correlated with the level of viral DNA methylation in the mouse genome.

Gas6/Axl is the sensor of arginine-auxotrophic response in targeted chemotherapy with arginine-depleting agents.

Many human malignancies lack de novo biosynthesis of arginine (Arg) as the key enzyme argininosuccinate synthetase 1 (ASS1) is silenced. These tumors acquire ectopic Arg for survival, and depleting this source by Arg-depleting recombinant enzyme ADI-PEG20 results in cell death. Mechanisms underlying Arg auxotrophy in these tumors and how they respond to Arg-auxotrophic stress are poorly understood. Here, we report that an immediate-early event of Arg-auxotrophic response involves reactive oxygen species-mediated secretion of Gas6, which interacts with its receptor Axl and activates the downstream Ras/PI3K/Akt growth signal leading to accumulation of c-Myc by protein stabilization. Arg-auxotrophic challenge also transcriptionally upregulates c-Myc expression, which provides a feedback mechanism to enhance Axl expression. c-Myc is a positive regulator of ASS1, but elevated ASS1 provides a feedback mechanism to suppress c-Myc and Axl. Our results revealed multiple inter-regulatory pathways in Arg-auxotrophic response, consisting of Axl, c-Myc and ASS1, which regulate Arg homeostasis and ADI-PEG20 sensitivity. These pathways provide potential targets for improving the efficacy of treating Arg-auxotrophic tumors using Arg-deprivation strategies.

Overexpression of gamma-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1.

Tumor necrosis factor (TNF) is a highly pleiotropic cytokine whose activity is at least partially regulated by the redox status of the cell. The cellular redox status is controlled primarily by glutathione, a major cellular antioxidant, whose synthesis is regulated by the rate-limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS). In the present report we investigated the effect of gamma-GCS overexpression on the TNF-induced activation of nuclear transcription factors NF-kappa B and AP-1, stress-activated protein kinase/c-Jun amino-terminal kinase (JNK) and apoptosis. Transfection of cells with gamma-GCS cDNA blocked TNF-induced NF-kappa B activation, cytoplasmic I kappa B alpha degradation, nuclear translocation of p65, and NF-kappa B-dependent gene transcription. gamma-GCS overexpression also completely suppressed NF-kappa B activation induced by phorbol ester and okadaic acid, whereas that induced by H2O2, ceramide, and lipopolysaccharide was minimally affected. gamma-GCS also abolished the activation of AP-1 induced by TNF and inhibited TNF-induced activation of JNK and mitogen-activated protein kinase kinase. TNF-mediated cytotoxicity and activation of caspase-3 were both abrogated in gamma-GCS-overexpressing cells. Overall, our results indicate that most of the pleiotropic actions of TNF are regulated by the glutathione-controlled redox status of the cell.

Effects of neocarzinostatin on mammalian nuclei: release of nucleosomes.

When Chinese hamster cell nuclei (Line CHO) were reacted with neocarzinostatin and its DNA was analyzed on non-denaturing agarose gel electrophoresis, a series of bands with a multiplicity of 175 base pairs was obtained. A similar result was also obtained when the DNA samples were electrophoresed under denaturing gels. Our results suggest that the linker DNA between nucleosomes is the susceptible site to the drug and that neocarzinostatin can be used to study the chromatin structure.