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Cancer Research Dec 1996Methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and methionine metabolism, is present in all normal tissues but is frequently deficient in a...
Methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and methionine metabolism, is present in all normal tissues but is frequently deficient in a variety of cancers. It has been suggested that this metabolic difference between normal and cancer cells may be exploited to selectively treat MTAP-negative cancers by inhibiting de novo purine synthesis and by depleting L-methionine. However, these therapeutic strategies have only been tested in naturally occurring MTAP-positive and -negative cell lines, which might have additional genetic alterations that affect chemotherapeutic sensitivity. Therefore, it is of importance to examine the feasibility of enzyme-selective treatment using paired cell lines that have an identical genotype except for MTAP status. MTAP-negative A549 lung cancer cells were transfected with eukaryotic expression vectors encoding MTAP cDNA in sense and antisense orientations. The resultant stable transfectomas were treated with inhibitors of de novo purine synthesis such as methotrexate, 5,10-dideazatetrahydrofolate, and L-alanosine and by methionine depletion. The A549 cells transfected with an antisense construct (antisense transfectoma) expressed no MTAP protein and were more sensitive to both purine and methionine depletion than were cells expressing MTAP protein (sense transfectoma). Methylthioadenosine was able to completely rescue the sense transfectoma but not the antisense transfectoma from growth inhibition by depletion of purine and methionine. These results prove that MTAP deficiency contributes directly to the sensitivity of cancer cells to purine or methionine depletion. Inhibition of de novo purine synthesis, combined with methionine depletion in the presence of methylthioadenosine, is a highly selective treatment for MTAP-negative cancers.
Topics: Alanine; Antimetabolites, Antineoplastic; Drug Resistance, Neoplasm; Feasibility Studies; Genetic Vectors; Humans; Lung Neoplasms; Methionine; Methotrexate; Neoplasm Proteins; Purine-Nucleoside Phosphorylase; Purines; Tetrahydrofolates; Transcription, Genetic; Transfection; Tumor Cells, Cultured
PubMed: 8971171
DOI: No ID Found -
Blood Cells, Molecules & Diseases 2002We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts...
We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.
Topics: Alanine; Antimetabolites, Antineoplastic; Cell Division; Chromosome Deletion; Chromosomes, Human, Pair 9; Cyclin-Dependent Kinase Inhibitor p16; Drug Resistance, Neoplasm; Humans; Leukemia-Lymphoma, Adult T-Cell; Neoplasm Proteins; Purine-Nucleoside Phosphorylase; Transfection; Trimetrexate; Tumor Cells, Cultured
PubMed: 11987241
DOI: 10.1006/bcmd.2002.0483 -
The Journal of Urology Feb 1984Two hundred six different samples of human renal carcinoma were tested for in vitro chemotherapy sensitivity using a soft agar colony formation assay similar to that... (Comparative Study)
Comparative Study
Two hundred six different samples of human renal carcinoma were tested for in vitro chemotherapy sensitivity using a soft agar colony formation assay similar to that originally described by Salmon and colleagues. Eighty of 159 (50 per cent) evaluable tumor tests showed colony formation in vitro and gave clinical drug sensitivity information. Two-thirds of tumors were resistant to all drugs tested, despite a median number of drugs tested per tumor of 14.5. Five tumors (6 per cent) were remarkably sensitive to numerous anticancer drugs in vitro. The most active drugs found in vitro were teniposide, actinomycin D, bleomycin, hydroxyurea, mitoguanazone dihydrochloride, mitomycin C and L-alanosine. Fourteen other drugs tested showed low in vitro cytotoxicity.
Topics: Adenocarcinoma; Agar; Antineoplastic Agents; Bleomycin; Cells, Cultured; Dactinomycin; Drug Evaluation; Humans; Hydroxyurea; Kidney Neoplasms; Mitoguazone; Nephrectomy; Teniposide
PubMed: 6199522
DOI: 10.1016/s0022-5347(17)50392-4 -
The International Journal of... Mar 1996The metabolism of adenine nucleotides (AdRN) has been studied previously in whole brains, brain slices and brain extracts, containing mixed populations of neurons and...
The metabolism of adenine nucleotides (AdRN) has been studied previously in whole brains, brain slices and brain extracts, containing mixed populations of neurons and glia. The availability of primary neuronal cultures enables us to study these pathways in almost pure neuronal preparations. The aim of the present study was to characterize the relative importance of the pathways of AdRN metabolism in the neurons. The metabolic fate of (8-14C) adenine and of AdRN prelabeled with (8-14C)adenine were studied in immature and mature primary rat neuronal cultures. Specific inhibitors were used to clarify the various metabolic fluxes, which were evaluated based on the time-related changes in the distribution of label (the cellular nucleotide content did not change during incubation). The turnover rate of AdRN was found to reflect mainly conversion of label to acid insoluble derivatives (AID) and partly degradation to hypoxanthine. The turnover was faster in the immature neurons. The combined addition of 2'-deoxycoformycin (2'-dCF) and of 5'-amino-5'-deoxyadenosine, inhibiting adenosine metabolism, resulted in both cultures in enhanced loss of label from AdRN, mainly to adenosine and adenine. This finding indicates the activity of the futile cycle AMP-->adenosine-->AMP. In both cultures, in the presence of these inhibitors, the ratio (hypoxanthine + inosine)/(adenine + adenosine) was 1.1, indicating that the fluxes through AMP deamination and AMP dephosphorylation are about equal. Addition of L-alanosine, inhibiting the conversion of IMP to AMP, resulted in both cultures, but especially in the mature neurons, in enhanced loss of label from AdRN to hypoxanthine and inosine. This finding indicates the functioning of the adenine nucleotide cycle (AMP-->IMP-->adenylosuccinic acid-->AMP). Under conditions of enhanced degradation of ATP (induced by iodoacetate and antimycin A), addition of 2'-dCF resulted in the immature cultures in lowering the ratio (hypoxanthine + inosine + IMP)/(adenine + adenosine) to 0.62, indicating a shift in favor of AMP dephosphorylation.
Topics: Adenine Nucleotides; Alanine; Animals; Cells, Cultured; Cellular Senescence; Homeostasis; Neurons; Purines; Rats
PubMed: 8920641
DOI: 10.1016/1357-2725(95)00134-4 -
The Journal of Biological Chemistry Jul 1983The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the...
The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.
Topics: Adenosine Deaminase; Animals; Cell Line; Coformycin; Electrophoresis, Polyacrylamide Gel; Electrophoresis, Starch Gel; Gene Amplification; Genes; Karyotyping; Mice; Molecular Weight; Nucleoside Deaminases; Pentostatin; Thymidine Kinase
PubMed: 6602803
DOI: No ID Found -
The American Journal of Surgical... Nov 2005The gene that encodes methylthioadenosine phosphorylase (MTAP), an enzyme involved in adenine and methionine salvage pathways, is located on chromosome 9p21 telomeric to...
Concordant loss of MTAP and p16/CDKN2A expression in gastroesophageal carcinogenesis: evidence of homozygous deletion in esophageal noninvasive precursor lesions and therapeutic implications.
The gene that encodes methylthioadenosine phosphorylase (MTAP), an enzyme involved in adenine and methionine salvage pathways, is located on chromosome 9p21 telomeric to the p16INK4A/CDKN2A tumor suppressor gene. Inactivation of the p16INK4A/CDKN2A gene occurs by three different mechanisms: hypermethylation of the gene promoter, intragenic mutation coupled with loss of the second allele, and homozygous deletion. Immunohistochemical labeling for the p16INK4A/CDKN2A gene product parallels gene status but does not elucidate the mechanism of gene inactivation. Since the MTAP gene is often co-deleted with p16INK4A/CDKN2A, concurrent immunolabeling for both proteins can identify cases with homozygous p16INK4A/CDKN2A gene deletion. MTAP loss itself has therapeutic implications since it may confer selective sensitivity to inhibitors of de novo purine biosynthesis, such as L-alanosine. Twelve tissue microarrays were constructed from 92 cases of Barrett-associated adenocarcinomas and precursor lesions and 112 cases of gastric adenocarcinoma and precursor lesions comprising 1161 individual cores. Multiple cores were arrayed from any given case, and when available, included the entire histologic spectrum of intestinal metaplasia-dysplasia-carcinoma. Tissue microarrays were labeled with monoclonal antibodies against MTAP protein (clone 6.9, Salmedix, Inc) and p16 (clone 16P07, Neomarkers). Complete loss of labeling was considered negative, while any labeling (p16: nuclear; MTAP: cytoplasmic and nuclear) was considered positive. Loss of MTAP labeling occurred exclusively in conjunction with loss of p16 labeling, confirming that the previous findings from this group that concurrent loss of MTAP and p16 labeling is a surrogate marker of 9p21 homozygous deletions. Complete loss of MTAP and p16 was seen in 4 of 25 (16%) patients with Barrett's esophagus, 4 of 18 (22%) with low-grade dysplasia, 5 of 39 (13%) with high-grade dysplasia, 17 of 78 (22%) with invasive adenocarcinoma, and 8 of 36 (22%) of metastases. There were 7 cases of esophageal adenocarcinoma with loss of both MTAP and p16 for which precursor lesions were available. In 6 on these 7 cases (85%), the precursor lesion(s) had loss of both MTAP and p16. Lack of MTAP and p16 expression was seen in 11 of 106 (10%) cases of gastric adenocarcinoma. All metaplastic (30 biopsies from 20 cases) and dysplastic (15 biopsies from 13 cases) gastric tissues had both intact MTAP and p16INK4A/CDKN2A gene products. No precursor lesions were available from the gastric cancers that had loss of both MTAP and p16. Two benign gastric hyperplastic polyps also had intact p16 and MTAP. Concurrent MTAP and p16 loss detected by immunohistochemistry can serve as a convenient surrogate for p16INK4A/CDKN2A gene homozygous deletion in archival tissues. Inactivation of p16INK4A/CDKN2A by homozygous deletion appears to be an early event in Barrett carcinogenesis, occurring in noninvasive precursor lesions, including nondysplastic Barrett mucosa, in subsets of cases. In the absence of MTAP, cells depend exclusively on the de novo synthesis pathway for production of adenosine. This loss of MTAP during 9p21 homozygous deletion might be exploited therapeutically using de novo purine synthesis antimetabolites to treat a subset of invasive gastroesophageal adenocarcinomas and esophageal precursor lesions.
Topics: Adenocarcinoma; Barrett Esophagus; Esophageal Neoplasms; Gene Deletion; Genes, p16; Humans; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Purine-Nucleoside Phosphorylase; Stomach Neoplasms
PubMed: 16224217
DOI: 10.1097/01.pas.0000170349.47680.e8 -
Clinical Cancer Research : An Official... Jun 2003Homozygous deletions at chromosome region 9p21 targeting the CDKN2A gene have been reported as a common cytogenetic abnormality in mesothelioma. MTAP, a gene...
PURPOSE
Homozygous deletions at chromosome region 9p21 targeting the CDKN2A gene have been reported as a common cytogenetic abnormality in mesothelioma. MTAP, a gene approximately 100-kb telomeric to CDKN2A, encodes methylthioadenosine phosphorylase, an enzyme essential in the salvage of cellular adenine and methionine, and its codeletion with CDKN2A has been reported in other tumors. The aim of this study was to define the prevalence of homozygous deletion of CDKN2A alone or in combination with MTAP in a large series of pleural mesothelioma.
EXPERIMENTAL DESIGN
We used a fluorescent in situ hybridization assay for CDKN2A and MTAP on interphase nuclei in imprints of frozen tissue from 95 cases of pleural mesothelioma. Histologically, the cases were classified as epithelial (71), biphasic (19) and sarcomatous (5). In each experiment, a 9p21 locus specific probe and a chromosome 9 centromeric probe were used and fluorescent in situ hybridization signals for both probes were simultaneously recorded in at least 100 nuclei. Cases were considered homozygously deleted if both 9p21 signals were lost in at least 20% of nuclei.
RESULTS
Overall, 70 cases (74%) had homozygous deletion of CDKN2A. MTAP was codeleted in 64 of these cases (91%). No case with MTAP deletion without CDKN2A deletion was identified. Homozygous loss of CDKN2A was seen in 49 of 71 epithelial (70%), 16 of 19 biphasic (89%), and 5 of 5 sarcomatous (100%) mesotheliomas.
CONCLUSIONS
Homozygous deletion of CDKN2A is seen in the majority of pleural mesotheliomas, and MTAP is codeleted in most of these cases. Previous cell line studies have shown that loss of MTAP renders cells dependent on de novo synthesis of purine derivatives. Thus, the particularly high prevalence of MTAP codeletion in mesothelioma makes it an ideal candidate for trials of targeted therapy using inhibitors of de novo AMP synthesis (e.g., L-alanosine).
Topics: Adult; Aged; Female; Gene Deletion; Genes, p16; Humans; In Situ Hybridization, Fluorescence; Male; Mesothelioma; Middle Aged; Pleural Neoplasms; Purine-Nucleoside Phosphorylase
PubMed: 12796375
DOI: No ID Found -
Biochimica Et Biophysica Acta Apr 1995GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The...
GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [14C]glycine (to label the de novo pathway) or [3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or L-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73-81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, L-alanosine decreased ATP mass by 26-33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.
Topics: Adenosine Triphosphate; Alanine; Animals; Cytosol; Guanosine Triphosphate; Insulin; Islets of Langerhans; Male; Mercaptopurine; Mycophenolic Acid; Rats; Rats, Sprague-Dawley
PubMed: 7718617
DOI: 10.1016/0167-4889(94)00235-7 -
Biochemical and Biophysical Research... May 1996Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation)....
Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation). NPM requires GTP for localization into nucleoli (J. Biol. Chem. 268, 5823-5827, 1993). To understand more about NPM's dynamic localization, the effects of lowering ATP on NPM-translocation and rRNA synthesis were studied. When the ATP level in HeLa cells was reduced by sodium azide, NPM-translocation was blocked. Similar results were obtained when ATP was depleted by other agents, suggesting that ATP depletion was responsible for the blocking of NPM-translocation. It was found that newly synthesized rRNA accumulated in the nuclei during ATP-depletion. Significantly larger than normal nucleoli were also observed. These results indicate that NPM may be involved in the transportation of newly synthesized ribosomes.
Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Alanine; Antibiotics, Antineoplastic; Azides; Cell Nucleus; Dinitrophenols; HeLa Cells; Humans; Kinetics; Mycophenolic Acid; Nuclear Proteins; Nucleolus Organizer Region; Nucleophosmin; RNA, Ribosomal; Ribosomes; Sodium Azide
PubMed: 8670243
DOI: 10.1006/bbrc.1996.0782 -
Biology of Reproduction Jun 1994This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from immature mice 48 h after priming...
This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from immature mice 48 h after priming with 5 IU eCG and culture for 3 h in medium containing 14C-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercaptopurine. Tissue extracts from complexes were analyzed by HPLC using either a C18 reversed-phase column (for separation of purine bases and nucleosides) or an ion exchange column (for separation of nucleotides). Most of the hypoxanthine taken up by complexes was salvaged to inosine monophosphate (IMP) and then converted to nucleotides. Metabolism favored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encountered, and metabolism to inosine via purine nucleoside phosphorylase was negligible. A similar pattern of hypoxanthine metabolism was observed in extracts of oocytes that had been denuded after the culture period. Addition of alanosine to the culture medium significantly reduced the synthesis of adenyl nucleotides in complexes and partially shunted metabolism in the direction of guanyl nucleotides. However, neither alanosine nor another inhibitor of adenylosuccinate synthetase, hadacidin, significantly influenced the meiotic arrest maintained by hypoxanthine. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotides but did not appreciably affect metabolism to other nucleotides. 6-Mercaptopurine produced an increase in the hypoxanthine-containing peaks, which was consistent with suppression of purine salvage. These results demonstrate that hypoxanthine is readily salvaged by the murine oocyte-cumulus cell complex and that the inhibitor-induced changes in metabolism are consistent with the presumed mechanism of action of each inhibitor. In addition, whereas metabolism favors conversion of IMP to adenyl nucleotides, synthesis of adenyl nucleotides by this route during the culture period is apparently not required for hypoxanthine-maintained meiotic arrest in vitro.
Topics: Adenylosuccinate Synthase; Alanine; Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Female; Hypoxanthine; Hypoxanthines; Inosine; Inosine Monophosphate; Meiosis; Mercaptopurine; Mice; Mycophenolic Acid; Oocytes; Purine-Nucleoside Phosphorylase; Purines; Uric Acid; Xanthine Oxidase
PubMed: 8080928
DOI: 10.1095/biolreprod50.6.1403