thioguanine-anhydrous and Cell-Transformation--Neoplastic

Colitis-associated cancer represents a long-standing problem, with two new factors adding to its importance: the diffusion of inflammatory bowel disease in developing countries, and the increased availability of effective drugs that control ulcerative colitis delaying or abrogating the need for a curative colectomy. The consolidated evidence that inflammation is the unique variable that factors in colitic cancer development has conferred impetus to the search and release of anti-inflammatory/immune suppressive molecules to pursue the goal of cancer chemoprevention. Cutting-edge research has provided breakthrough insights into the mechanism of the chemopreventive actions of mesalamines, thiopurines, and probiotics, and we expand on these topics. Despite these advancements, bedside evidence is still mixed and calls for further scrutiny. Nowadays, the clinician must continue to rely on classic preventive measures such as surveillance colonoscopy, and the early and aggressive use of drugs that permit to keep the degree of mucosal inflammation to a minimum.

Topics: Animals; Anti-Inflammatory Agents; Cell Transformation, Neoplastic; Chemoprevention; Colonoscopy; Colorectal Neoplasms; Humans; Inflammatory Bowel Diseases; Mesalamine; Probiotics; Thioguanine

Cancer appears to be a disease of altered maturation, with changes in genetic expression leading to a situation in which the physiological regulation of cellular proliferation and maturation are altered. Environmental factors as well as defined chemical agents have been demonstrated to have the capacity to convert neoplastic cells to end-stage forms with a finite life span through a process characteristic of cellular maturation. The correction of genetic defects by these inducers of differentiation does not appear to be required; the critical feature is that the differentiated cells assume a state in which they no longer possess the capability for continued cellular replication. The extrapolation of these advances, accomplished in experimental systems, to clinical practice should yield significant decreases in the neoplastic cell burden without the degree of morbidity produced by aggressive therapy with cytodestructive agents, especially when employed in multidrug combinations. The ultimate introduction of differentiation as a therapeutic approach to cancer treatment if attained, however, will require a variety of principles to be established, so that optimum efficacy may be obtained from each agent, the fabrication of new agents with major changes in the ratio of the concentrations required to produce cytotoxicity relative to those necessary to initiate maturation is attained, and the elucidation of non-antagonistic combinations of differentiation inducing agents with or without cytotoxic drugs is achieved to combat the problem of tumor cell heterogeneity.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Humans; Hydrocortisone; Leukemia, Experimental; Models, Biological; Naphthacenes; Phenotype; Thioguanine; Tretinoin

Most drugs available for cancer chemotherapy exert their effects through cytodestruction. Although significant advances have been attained with these cytotoxic agents in several malignant diseases, response is often accompanied by significant morbidity and many common malignant tumours respond poorly to existing cytotoxic therapy. Development of chemotherapeutic agents with non-cytodestructive actions appears desirable. Considerable evidence exists which indicates that (a) the malignant state is not irreversible and represents a disease of altered maturation, and (b) some experimental tumour systems can be induced by chemical agents to differentiate to mature end-stage cells with no proliferative potential. Thus, it is conceivable that therapeutic agents can be developed which convert cancer cells to benign forms. To study the phenomenon of blocked maturation, squamous carcinoma SqCC/Y1 cells were employed in culture. Using this system it was possible to demonstrate that physiological levels of retinoic acid and epidermal growth factor were capable of preventing the differentiation of these malignant keratinocytes into a mature tissue-like structure. The terminal differentiation caused by certain antineoplastic agents was investigated in HL-60 promyelocytic leukaemia cells to provide information on the mechanism by which chemotherapeutic agents induce cells to by-pass a maturation block. The anthracyclines aclacinomycin A and marcellomycin were potent inhibitors of N-glycosidically linked glycoprotein biosynthesis and transferrin receptor activity, and active inducers of maturation; temporal studies suggested that the biochemical effects were associated with the differentiation process. 6-Thioguanine produced cytotoxicity in parental cells by forming analog nucleotide. In hypoxanthine-guanine phosphoribosyltransferase negative HL-60 cells the 6-thiopurine initiated maturation; this action was due to the free base (and possibly the deoxyribonucleoside), a finding which separated termination of proliferation due to cytotoxicity from that caused by maturation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Leukemia, Myeloid; Models, Biological; Naphthacenes; Thioguanine; Tretinoin

An uncompromisingly optimistic approach must now be taken to the future of the treatment of acute myelogenous leukemia. This view can be justified on two grounds. First, understanding of the biology of the disease is increasing, resulting for example in the demonstration of the prognostic significance of the behavior of the leukemic blast cells in culture and their chromosomal pattern. It seems most likely that individualization of treatment will follow from such observations, with obvious benefit to the patients. Second it has been shown that a modest proportion of patients is cured with the manipulations of chemotherapy with or without radiotherapy and bone marrow transplantation practised in the mid-1970s. Selected results reflecting a personal bias have been presented allowing speculation that very intensive chemotherapy, possibly of short duration may be able to increase this proportion. At the same time, advances in the techniques of preparation of both patient and bone marrow for transplantation are being made and may increase the potential pool of patients who may benefit from the procedure. Even within the limitations of the treatment available now, it is possible that a flexible attitude to the precise manipulation of cytotoxic drugs, radiotherapy and transplantation may result in cure for the majority, rather than the minority, of patients.

Topics: Adolescent; Adult; Antibiotics, Antineoplastic; Bone Marrow; Cell Transformation, Neoplastic; Cytarabine; Drug Therapy, Combination; Humans; Leukemia, Myeloid, Acute; Middle Aged; Naphthacenes; Prednisolone; Prognosis; Regression Analysis; Thioguanine; Time Factors; Vincristine

In this prospective randomized multicenter trial 93 patients, median age 72 years, with RAEB-t (n=25) and myelodysplastic syndrome (MDS)-AML (n=68) were allocated to a standard induction chemotherapy regimen (TAD 2+7) with or without addition of granulocyte-macrophage-CSF (GM-CSF). The overall complete remission (CR) rate was 43% with no difference between the arms. Median survival times for all patients, CR patients, and non-CR patients were 280, 550, and 100 days, respectively, with no difference between the arms. Response rates were significantly better in patients with serum lactate dehydrogenase (S-LDH) levels

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Anemia, Refractory, with Excess of Blasts; Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Cytarabine; Daunorubicin; Female; Follow-Up Studies; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Prospective Studies; Remission Induction; Survival Rate; Thioguanine

Thirty-two patients in the blastic phase of Philadelphia chromosome-positive chronic granulocytic leukemia (CGL) were studied in a prospective randomized trial in which vincristine--prednisone (19 patients) was compared with cytosine arabinoside--6-thioguanine (13 patients). Seven remissions (37%), including two complete remissions, were achieved in the vincristine--prednisone group. Three of the five with predominant hypodiploid blast cell lines treated with vincristine--prednisone had complete or partial remissions. Both complete remitters presented with hypodiploidy consisting of 44 chromosomes. Four patients (30%) who were treated with cytosine arabinoside--6-thioguanine responded with one complete remission. The median survival of the responders was 8 mo, as compared to 1--2 mo for the nonresponders. Crossover to the opposite regimen as secondary therapy following refractoriness or resistance resulted in only 3 partial responses out of 21 treated. All three had previously responded to vincristine--prednisone. Of the 32 cases, 14 had an elective splenectomy during the chronic phase of the disease. Prior splenectomy did not influence the response to chemotherapy, as all three complete remitters occurred in the nonsplenectomized group. Similarly, survival in the blastic phase was not affected by prior splenectomy.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Child; Chromosomes, Human, 21-22 and Y; Cytarabine; Diploidy; Drug Therapy, Combination; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Prednisone; Remission, Spontaneous; Thioguanine; Vincristine

The relationships between telomerase and telomeres represent attractive targets for new anticancer agents. Here, we report that the nucleoside analogue 6-thio-2'-deoxyguanosine (6-thio-dG) is recognized by telomerase and is incorporated into de novo-synthesized telomeres. This results in modified telomeres, leading to telomere dysfunction, but only in cells expressing telomerase. 6-Thio-dG, but not 6-thioguanine, induced telomere dysfunction in telomerase-positive human cancer cells and hTERT-expressing human fibroblasts, but not in telomerase-negative cells. Treatment with 6-thio-dG resulted in rapid cell death for the vast majority of the cancer cell lines tested, whereas normal human fibroblasts and human colonic epithelial cells were largely unaffected. In A549 lung cancer cell-based mouse xenograft studies, 6-thio-dG caused a decrease in the tumor growth rate superior to that observed with 6-thioguanine treatment. In addition, 6-thio-dG increased telomere dysfunction in tumor cells in vivo. These results indicate that 6-thio-dG may provide a new telomere-addressed telomerase-dependent anticancer approach.. Telomerase is an almost universal oncology target, yet there are few telomerase-directed therapies in human clinical trials. In the present study, we demonstrate a small-molecule telomerase substrate approach that induces telomerase-mediated targeted "telomere uncapping," but only in telomerase-positive cancer cells, with minimal effects in normal telomerase-negative cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Deoxyguanosine; DNA Damage; Epithelial Cells; Female; Fibroblasts; Heterografts; Humans; Kidney; Liver; Mice; Substrate Specificity; Telomerase; Telomere; Telomere Homeostasis; Telomere Shortening; Thioguanine; Thionucleosides; Tumor Burden; Xenograft Model Antitumor Assays

Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine ((S)G) or S⁶-methylthioguanine (S⁶mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both (S)G and S⁶mG were mutagenic, resulting in G→A mutation at frequencies of ~8% and ~39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of (S)G and S⁶mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Topics: Antineoplastic Agents; Base Sequence; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA; Genetic Vectors; Humans; Immunosuppressive Agents; Molecular Sequence Data; Mutagenesis; Mutagens; Thioguanine

The initial processes involved in radiation carcinogenesis have not been clearly elucidated. We isolated mouse mutant cells exhibiting plasticity in their mutation phenotypes. These mutant cells were originally isolated from an irradiated cell population as 6-thioguanine resistant (6TGR) mutants that were deficient in hypoxanthine phosphoribosyl transferase (Hprt, E.C.2.4.2.8) activity at the frequency of approximately 6.2 x 10(-5). Approximately 10% of 6TGR cells showed plasticity in their mutant phenotypes and reverted to HAT-resistant (HATR), which is Hprt-proficient, wild type phenotype. Eventually we identified the plastic mutants in the un-irradiated wild type cell population as well and found that ionizing irradiation enhanced the frequency of the plastic mutation approximately 24 times. Treatment with 5-aza-cytidine did not affect the plasticity of mutant phenotypes identified in this study, suggesting that DNA methylation was not involved in the plastic changes of the mutant phenotypes. The plastic mutant phenotype identified in our study is a new type of genomic instability induced by ionizing irradiation, and it is likely to be involved in one of the primary changes that occur in the process of radiation carcinogenesis, and may explain one element of carcinogenesis, which is composed of multi-stages.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Genomic Instability; Hypoxanthine Phosphoribosyltransferase; Loss of Heterozygosity; Mice; Polymerase Chain Reaction; Thioguanine; X-Rays

A transformation assay using BALB/c 3T3 cells was conducted on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to assess initiation and promotion activities of MX carcinogenesis. Statistically significant positive responses were obtained compared with the corresponding solvent controls in both the initiation assay post-treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) and the promotion assay pretreated with 3-methylcholanthrene (MCA). Both TPA and MX inhibited metabolic cooperation in an assay using co-culture of V79 6-thioguanine (6-TG) sensitive and insensitive cells. However, cells isolated from transformed foci in the initiation assay did not induce any nodules after inoculation to BALB/c mice, the strain of mouse from which the transformation assay cells were derived. Although the study was carried out for 2-3 weeks, this might have been too short to develop nodules under the conditions of this experiment. This in vitro cell transformation study with MX adds supportive information to studies showing MX carcinogenicity and tumour promoter activity, and adds mechanistic understanding of the action of MX.

Topics: Animals; BALB 3T3 Cells; Biological Assay; Carcinogens; Cell Transformation, Neoplastic; Coculture Techniques; Furans; Mice; Tetradecanoylphorbol Acetate; Thioguanine

A patient is described with myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML) FAB M4. Cytogenetic analysis revealed an unusual rearrangement between chromosomes 9 and 17, leading to a dicentric chromosome with an insertion of material of unknown origin between both chromosomes. By fluorescence in situ hybridization (FISH), the insertion was shown to be an amplification of part of 17q, involving ERBB2, RARA, and TOP2A genes. The median copy number of ERBB2, RARA, and TOP2A genes in the tumor cells was six (range: 4--10). Only one copy of the MPO gene at 17q21.3 was detected, suggesting a deletion of the telomeric part of 17q. To our knowledge, this is the first report of a 17q amplification in AML.

Topics: Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Mapping; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 9; Cytarabine; Daunorubicin; DNA Topoisomerases, Type II; DNA-Binding Proteins; Female; Genes, erbB-2; Humans; In Situ Hybridization, Fluorescence; Isoenzymes; Karyotyping; Leukemia, Myelomonocytic, Acute; Metaphase; Middle Aged; Myelodysplastic Syndromes; Poly-ADP-Ribose Binding Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Thioguanine

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.

Topics: Animals; Cell Cycle; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; DNA-Binding Proteins; Drug Resistance; Gene Deletion; Mice; Phenotype; Radiation Tolerance; Skin Neoplasms; Thioguanine; Tumor Cells, Cultured; Ultraviolet Rays; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group A Protein

Growth factors have been reported to enhance the cytotoxicity of anticancer agents. In our study we investigated the capacities of interleukin 3 (IL-3), interleukin 7 (IL-7), low-molecular-weight B-cell growth factor (lmw-BCGF), and IL-3 + 7 to induce proliferation and to modulate the drug resistance of childhood acute lymphoblastic leukemia (ALL) cells. Proliferation was assessed with the methyl-thiazole-tetrazolium (MTT) assay and other parameters. Cellular resistance to cytarabine, thioguanine, and prednisolone was measured using the MTT assay. In 19 samples containing >90% leukemic cells the proliferative response and the modulation of drug resistance was markedly heterogeneous between patient samples and between growth factors. All growth factors were able to stimulate proliferation significantly after 5 days of culture. lmw-BCGF was the most potent growth factor in this respect. Cytotoxicity of cytarabine and thioguanine was significantly increased by IL-7, that of thioguanine by IL-3 as well. IL-7 enhanced the cytotoxicity of thioguanine significantly more than IL-3 and lmw-BCGF and that of cytarabine more than IL-3. Cytotoxicity of prednisolone was not significantly influenced by any growth factor. In individual cases, growth factors reduced the cytotoxicity of the drugs. IL-3 + 7 did not add activity to the most potent single growth factor in both proliferation and drug resistance measurements. This study shows that IL-3, IL-7, and lmw-BCGF generally induce and occasionally inhibit proliferation of ALL cells. Furthermore, they may either increase or decrease cytotoxicity of anticancer drugs. This heterogeneous response to growth factors concerning induction of proliferation and modulation of drug resistance should be taken into account in their clinical use.

Topics: Cell Count; Cell Transformation, Neoplastic; Child; Child, Preschool; Cytarabine; Drug Resistance, Neoplasm; Humans; Interleukin-3; Interleukin-7; Lymphokines; Mitotic Index; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prednisolone; Thioguanine

Anchorage independence and gene amplification have frequently been associated with a transformed or tumorigenic phenotype in cultured mammalian cells. However, it is unknown whether these two traits occur as related events during transformation, or are independent features of the transformed phenotype. To clarify this point, immortalized, untransformed CHEF18 Chinese hamster cells were propagated in culture until they became transformed and tumorigenic. The frequencies with which CHEF18 cells formed colonies either in soft agar, in medium containing N-phosphonacetyl-L-aspartate or in the two selective media simultaneously, were determined. The results indicate that anchorage independence and CAD gene amplification spontaneously arose during the propagation of the cells and that their concurrent emergence was not the consequence of independent events. However, the kinetics of their appearance suggests that anchorage independence is the early event whereas gene amplification might represent one of the numerous events which can be dynamically selected in anchorage-independent cells.

Topics: Animals; Aspartate Carbamoyltransferase; Aspartic Acid; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Dihydroorotase; Drug Resistance; Gene Amplification; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Phosphonoacetic Acid; Thioguanine

A variety of purine analogs inhibit the growth and induce the differentiation of human promyelocytic leukemia (HL-60) cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mechanisms by which purine analogs induce differentiation offer unique potential for cancer chemotherapy. The guanine analogs, 6-thioguanine and 8-azaguanine, induce granulocytic differentiation of HGPRT-deficient HL-60 promyelocytes. Although these compounds are useful as model purine analogs that induce differentiation in HGPRT-deficient HL-60 cells, they suffer the disadvantage that they are highly cytotoxic to wild-type cells. We studied the effect of the hypoxanthine analog 6-ethylmercaptopurine on wild-type and HGPRT-deficient HL-60 cells. 6-Ethylmercaptopurine inhibits growth and produces a specific terminal end-cell in both types of HL-60 cells. The mechanism appears to be independent of the normal modes of cytotoxic activation through HGPRT or adenine phosphoribosyltransferase (APRT), since no new peaks were seen in HPLC chromatograms of the nucleotide pools. Furthermore, hypoxanthine and adenine failed to prevent growth inhibition by 6-ethylmercaptopurine, and inhibition of IMP dehydrogenase and the consequential alteration of the guanine nucleotide pools does not appear to be involved. The mechanism differs from that of guanine analog-induced differentiation in HGPRT-deficient HL-60 cells.

Topics: Antimetabolites, Antineoplastic; Cell Transformation, Neoplastic; Depression, Chemical; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Leukemia, Promyelocytic, Acute; Mercaptopurine; Thioguanine; Tumor Cells, Cultured

The cytotoxicity, mutagenicity and transforming potentials of three second generation platinum compounds have been investigated in mammalian cells. All the compounds showed positive response in two assay systems in Chinese hamster V 79 cells, i.e. measurement of mutation induction at the HGPRT locus and of DNA damage as indicated by sister chromatid exchange frequencies. At equitoxic doses, the compounds in order of decreasing mutagenicities were cisplatin, spiroplatin, carboplatin and iproplatin. The BHK transformation assay reflected a similar order in the potential carcinogenicity of the drugs. Cisplatin was highly carcinogenic, followed by spiroplatin. In comparison, carboplatin and iproplatin were potentially weak carcinogens.

Topics: Animals; Antineoplastic Agents; Carboplatin; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cisplatin; Cricetinae; DNA Damage; Mutagens; Organoplatinum Compounds; Sister Chromatid Exchange; Thioguanine

The genomic stability of a series of nontumorigenic, tumorigenic, and tumor-derived Chinese hamster embryo fibroblastic (CHEF) cell lines was compared by examining their rates of spontaneous mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, using thioguanine resistance for selection of mutants. The spontaneous mutation rates were 1.1 x 10(-6) mutations/cell/generation in the non-tumor-forming CHEF/18 cell line and 4.9 x 10(-6) in the tumorigenic CHEF/16 cells. Three tumorigenic and tumor-derived CHEF cell lines derived from CHEF/18 (J132 3-2 T3L, focus 2, focus 3) and two lines (16-2 Tuk 4 and 204 Bu50 Tuk 2) derived from CHEF/16 were chosen on the basis of their karyotypes, which demonstrated a considerable level of chromosomal rearrangement. Mutation rates of four of these five lines ranged from 1.2 x 10(-6) to 8.9 x 10(-6) mutations per cell per generation. Only the fifth line, 16-2 Tuk 4, showed a significantly elevated rate of mutation as compared with the nontumorigenic CHEF/18 cell line. Thus, we have found no simple correlation between spontaneous mutation rate and the malignant phenotype, and we conclude that mutation rate per se is not a sensitive index of malignancy. In addition, we have compared three methods of calculating mutation rate and find that they rank the cell lines in the same order, but each stresses a different aspect of the distribution and therefore produces different estimates of the mutation rate.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Drug Resistance; Embryo, Mammalian; Fibroblasts; Hypoxanthine Phosphoribosyltransferase; Mutation; Thioguanine; Translocation, Genetic

Three laboratories participated in an interlaboratory study to evaluate the usefulness of the Chinese hamster V79 cell metabolic cooperation assay to predict the tumor-promoting activity of selected chemicals. Twenty-three chemicals of different chemical structures (phorbol esters, barbiturates, phenols, artificial sweeteners, alkanes, and peroxides) were chosen for testing based on in vivo promotion activities, as reported in the literature. Assay protocols and materials were standardized, and the chemicals were coded to facilitate unbiased evaluation. A chemical was tested only once in each laboratory, with one of the three laboratories testing only 15 out of 23 chemicals. Dunnett's test was used for statistical analysis, and differences between treated- and control-cell responses were analyzed at P less than or equal to .01. Chemicals were scored as positive (at least two concentration levels statistically different than control), equivocal (only one concentration statistically different), or negative. For 15 chemicals tested in all three laboratories, there was complete agreement among the laboratories for nine chemicals. For the 23 chemicals tested in only two laboratories, there was agreement on 16 chemicals. With the exception of the peroxides and alkanes, the metabolic cooperation data were in general agreement with in vivo data. However, an overall evaluation of the V79 cell system for predicting in vivo promotion activity was difficult because of the organ specificity of certain chemicals and/or the limited number of adequately tested nonpromoting chemicals.

Topics: Animals; Carcinogens; Cell Communication; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Cricetinae; Cricetulus; Fibroblasts; Intercellular Junctions; Lung; Male; Predictive Value of Tests; Thioguanine

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2cm2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thio-guanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.

Topics: Animals; Azaguanine; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cricetulus; Guanine; Pentosyltransferases; RNA, Transfer, Amino Acid-Specific; RNA, Transfer, Amino Acyl; Thioguanine

The genotoxicity of soluble and insoluble hexavalent chromium compounds was studied in mammalian cell assays which detect base substitution, deletion, addition, and frameshift mutations [6-thioguanine resistance in Chinese hamster ovary cells], primarily base substitution mutations [ouabain resistance in Chinese hamster ovary and C3H/10T1/2 Cl 8 mouse embryo fibroblasts (10T1/2)] and morphological transformation [focus formation] in 10T1/2 cells. Soluble hexavalent CaCrO4, administered in either acute (5-h) or subacute (24-h) dosing regimens, induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance in Chinese hamster ovary cells but no mutation to ouabain resistance or focus formation in transformation assays, although the acute treatment induced a high frequency of conversion of 10T1/2 cells to adipocytes. Cell lines established from cloned adipocytic cells were not morphologically transformed and did not grow in soft agarose. PbCrO4 did not induce mutation to either 6-thioguanine or ouabain resistance but did induce a reproducible dose-dependent, low frequency of focus formation in 10T1/2 cells. Cell lines established from PbCrO4-induced foci stably formed foci when coseeded with 10T1/2 cells, had 3-5-fold increased saturation densities relative to nontransformed 10T1/2 cells, and formed colonies in soft agarose, indicating their likelihood to be neoplastic. Long term exposure of 10T1/2 cells to either CaCrO4 or PbCl2, even at 85% cytotoxic concentrations, or pretreatment of cells with either CaCrO4 or PbCl2 followed by treatment with the alternate compound, did not induce morphological transformation. Treatment of cells with insoluble hexavalent PbCrO4 resulted in progressive and extensive vacuolization of cells in contact with the particles. Progressive cytoplasmic engulfment of PbCrO4 particles was observed using scanning electron microscopy, although PbCrO4 particles were not observed inside vacuoles. These results indicate that the soluble clastogens K2Cr2O7 and CaCrO4 were probably mutagenic by a non-base substitution mechanism but could not transform 10T1/2 cells. In contrast, PbCrO4 was not detectably mutagenic but induced transformation, which could not be explained solely by acute or chronic exposure to dissolution products of either lead or chromate alone. Since PbCrO4 particles were found to be intracytoplasmic in extensively vacuolated cells, we suggest that the unique physiochemical properties of PbCrO4 particles, leadi

Topics: Adipose Tissue; Animals; Calcium Compounds; Cell Transformation, Neoplastic; Cells, Cultured; Chromates; Lead; Mice; Mice, Inbred C3H; Microscopy, Electron; Mutagenicity Tests; Ouabain; Thioguanine

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.

Topics: 4-Nitroquinoline-1-oxide; Adenine Phosphoribosyltransferase; B-Lymphocytes; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Child, Preschool; Colony-Forming Units Assay; Drug Resistance; Fluorenes; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Methylnitronitrosoguanidine; Mutagenicity Tests; Thioguanine

The changes in the frequencies of X-ray-induced mutations to 6-thioguanine resistance, chromosomal abnormalities and transformation to anchorage-independent growth were examined during confluent holding recovery in density-inhibited cultures of human diploid fibroblasts. Chromosomal abnormalities studied included non-stable aberrations, deletions, and reciprocal translocations measured up to 20 mean population doublings post-irradiation. Complex recovery kinetics were observed. A comparison of these kinetics suggests that reciprocal translocations are associated with the induction of both transformation and mutations. Mutagenesis correlated with deletions and translocations, whereas transformation correlated with translocations only.

Topics: Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes; Drug Resistance; Fibroblasts; Humans; Karyotyping; Mutation; Thioguanine

The relationship between the structure and genotoxic potential of four new cytostatic compounds--the ketonucleosides KN-35, KN-43, KN-44 and KN-3--was investigated in short-term in vitro assays of hypoxanthine-guanine phosphoribosyl transferase locus mutation in V79 cells, induction of chromosomal aberrations and sister chromatid exchanges on human lymphocytes, induction of chromosomal aberrations and micronuclei in V79 cells, and transformation of Syrian hamster embryo cells. None of the ketonucleosides induced mutagenic effects in any of the assays. Their failure to exhibit significantly genotoxic activity may be ascribed to the probable absence of any reaction between these drugs and the cellular DNA, and indicates that they act by some other mechanism which probably differs from the one observed with alkylating or intercalating antitumoural agents. This suggests that the cytotoxic activity of ketonucleosides cannot be related to genotoxicity.

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Chromosome Aberrations; Cricetinae; Drug Resistance; Humans; In Vitro Techniques; Mesocricetus; Mutagenicity Tests; Mutagens; Nucleosides; Sister Chromatid Exchange; Thioguanine

Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. A linear dose-dependence with a slope of approximately 1 was observed with both compounds when the data were plotted on a log-log graph. The trivalent sodium arsenite was greater than 10-fold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at non-toxic as well as toxic doses. At low doses, enhanced colony-forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges were induced by the arsenicals with similar dose-responses. These results support a possible role for chromosomal changes in arsenic-induced transformation.

Topics: Animals; Arsenates; Arsenic; Arsenites; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Cricetinae; Drug Resistance; Mutation; Ouabain; Sister Chromatid Exchange; Sodium Compounds; Thioguanine

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Humans; Lesch-Nyhan Syndrome; Mutation; Skin Neoplasms; Skin Physiological Phenomena; Sodium-Potassium-Exchanging ATPase; Thioguanine

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01-10 micrograms/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N'-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5-7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.

Topics: Animals; Benzo(a)pyrene; Benzopyrenes; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Diethylstilbestrol; Drug Resistance; Mesocricetus; Methylnitronitrosoguanidine; Mutation; Thioguanine; Time Factors

If neoplastic transformation of diploid human cells results from carcinogen-induced mutations, cells deficient in excision repair of UV-induced DNA damage should be significantly more sensitive to transformation by UV light than normal cells. We tested this hypothesis by irradiating fibroblasts from a xeroderma pigmentosum patient (XP7BE, complementation group D) with low doses of Uv light (254 nm) and cells from a normal person with much higher doses and comparing the frequency of transformation to anchorage independence. Both sets of cells exhibited a dose-dependent increase in transformation which corresponded to a dose-dependent decrease in survival. At doses that caused equal cell killing, the frequency of anchorage-independent cells was approximately equal. Colonies of XP7BE and normal cells isolated from agar, propagated, and injected into X-irradiated athymic mice produced fibrosarcomas in 100% of the animals. Normal cells irradiated shortly before the onset of DNA synthesis exhibited a high frequency of anchorage-independent cells; cells irradiated in early G1 showed no increase over background. These results agree with those we observed for UV induction of 6-thioguanine-resistant mutants in these cells and support the hypothesis that anchorage independence results from mutations induced by DNA replication on a damaged template.

Topics: Cell Cycle; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; Dose-Response Relationship, Radiation; Humans; Infant, Newborn; Male; Skin; Thioguanine; Ultraviolet Rays; Xeroderma Pigmentosum

We have demonstrated a dose-dependent increase in the frequency of diploid human cells capable of anchorage-independent (AI) growth after treatment with the carcinogen propane sultone, followed by exponential growth to allow full expression of this phenotype (8 to 13 population doublings). Exposure to these same concentrations of propane sultone also resulted in a dose-dependent increase in the frequency of 6-thioguanine-resistant cells in the population. Procedures such as synchronization of cells and treatment just after the onset of DNA synthesis or the use of special selective medium were not essential for this induction. A very low frequency of cells with the AI phenotype was found in the control population (background). Cells which exhibited the AI phenotype spontaneously or after carcinogen treatment retained the characteristic over as many generations as tested (greater than 13). The data suggest that AI growth is the result of a mutational event.

Topics: Anthralin; Cell Adhesion; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Drug Resistance; Fibroblasts; Humans; Methylnitronitrosoguanidine; Mutation; Neoplasms, Experimental; Thioguanine; Thiophenes

The ploidy dependence of the induced frequency of a phenotype can be used to determine the dominant or recessive nature of a somatic mutation to a given trait. To demonstrate this we induced mutations in diploid and spontaneously occurring tetraploid clones of Syrian hamster embryo cells by treatment with EMS (1.2 mg/ml, 4 h). Mutagenized cells were assayed for the recessive mutation to 6-thioguanine resistance (5 micrograms/ml) and the dominant mutation to ouabain resistance (1.2 mM). The frequency of induction of the dominant mutation was equal in the diploid and tetraploid clones (2.3 x 10(-4)). The frequency of induction of the recessive mutation was greatly reduced in the tetraploid clone relative to the diploid clone (1.8 x 10(-4) vs. 1.2 x 10(-3)). 6TGr mutant subclones from the tetraploid clone remain nearly tetraploid, or even increase in ploidy, but show a reduction in the number of X chromosomes from two to one, or in some cases none (based on chromosome morphology). The principle of ploidy dependence is now being used to study the induction of phenotypes related to neoplastic transformation.

Topics: Aneuploidy; Animals; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Drug Resistance; Embryo, Mammalian; Ethyl Methanesulfonate; Gene Frequency; Genes, Dominant; Genes, Recessive; Hypoxanthine Phosphoribosyltransferase; Mesocricetus; Mutagens; Ouabain; Phenotype; Thioguanine; Time Factors

A recent epidemiological survey in China showed that there is a regional distribution of esophageal cancer and a correlation between mortality for this cancer and environmental factors, especially the consumption of pickled vegetables. A series of experimental studies with pickled vegetable extract were done using different in vitro biological systems. The results showed that pickled vegetable extract induced 6-thioguanine-resistant mutants in V79 cells and increased sister chromatid exchanges in the same cells and in Syrian hamster embryo cells. Pickled vegetables extract induced transformed foci in Syrian hamster embryo cells and in 3-methylcholanthrene initiated C3H/10T1/2 cells. The mutagenic, transforming and promoting activities of pickled vegetable extract seen in vitro conform with in vivo results and provide evidence for the presence of a mutagen and/or a carcinogen in pickled vegetable extract. A possible role of pickled vegetables consumption in the etiology of esophageal cancer is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; China; Cricetinae; Cricetulus; Drug Resistance; Esophageal Neoplasms; Fibroblasts; Food Preservation; Food Preservatives; Male; Mesocricetus; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Mutagenesis; Mutagenicity Tests; Rats; Rats, Wistar; Sister Chromatid Exchange; Spices; Thioguanine; Vegetables

Thirty-seven patients with Philadelphia-chromosone-positive (Ph'+) chronic myelogenous leukemia who were untreated or minimally pretreated were entered on the L-5 protocol. This protocol consisted of sequential treatment with splenic irradiation, splenectomy, arabinosylcytosine and 6-thioguanine, and L-asparaginase. Maintenance therapy was hydroxyurea or a multiple-drug regimen. The median survival of the 37 patients is 50 mo. Twelve patients showed a temporary reduction in the percentage of Ph'+ marrow metaphases to less than one-third of the initial values and in 7 of these patients none were found. The duration of the Ph'+ chromosome reduction ranged from 1 to 43 mo. The median survival of the responders has not yet been reached. It is concluded that whereas overall survival is not appreciably extended, patients who have a reduction in Ph'+ cells in the marrow may survive longer than the average; also, the reduction occurs most frequently in patients who have relatively small spleens at diagnosis. The reduction is difficult to maintain, and it may be reinduced in some patients with intensive chemotherapy.

Topics: Adolescent; Adult; Asparaginase; Cell Transformation, Neoplastic; Chromosomes, Human, 21-22 and Y; Cytarabine; Female; Humans; Hydroxyurea; Leukemia, Myeloid; Male; Middle Aged; Radiography; Spleen; Splenectomy; Thioguanine

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.

Topics: Benzopyrenes; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Fructose; Genes; Mutation; Neoplasms, Experimental; Ouabain; Phenotype; Thioguanine; Urethane

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 X 10(-6) M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC or MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.

Topics: Animals; Azaguanine; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes; Cricetinae; Dose-Response Relationship, Drug; Drug Resistance; Guanidines; Mesocricetus; Methylguanidine; Methylnitronitrosoguanidine; Mutation; Nitrosamines; Thioguanine

Topics: Antibodies, Neoplasm; Antibody Formation; Antigen-Antibody Complex; Antigens, Neoplasm; BCG Vaccine; Cell Transformation, Neoplastic; Cytarabine; Daunorubicin; Drug Therapy, Combination; Humans; Immunologic Deficiency Syndromes; Immunotherapy; Leukemia, Myeloid, Acute; Remission, Spontaneous; Thioguanine

Topics: Animals; Cell Fusion; Cell Line; Cell Transformation, Neoplastic; Chromosomes; Gammaretrovirus; Haplorhini; Hybrid Cells; Kidney; Mice; Mice, Inbred C3H; Moloney murine leukemia virus; Parainfluenza Virus 1, Human; Pentosyltransferases; Rats; Retroviridae; Thioguanine; Virus Replication

Topics: Animals; Azaguanine; Cattle; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Culture Media; Diploidy; Drug Resistance; Glucosephosphate Dehydrogenase; Humans; Hypoxanthines; Immunosuppression Therapy; Isoenzymes; Karyotyping; L-Lactate Dehydrogenase; Methylcellulose; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Pentosyltransferases; Phenotype; Rabbits; Rats; Thioguanine; Thymus Gland

Topics: Acetyltransferases; Animals; Bromodeoxyuridine; Carbon Radioisotopes; Catechol O-Methyltransferase; Cattle; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Choline; Clone Cells; Enzyme Activation; Humans; Mercaptopurine; Mice; Neuroblastoma; Purines; Thioguanine; Time Factors; Tritium; Tyrosine 3-Monooxygenase

Topics: Adult; Age Factors; Antineoplastic Agents; Cell Transformation, Neoplastic; Cytarabine; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Prognosis; Remission, Spontaneous; Thioguanine; United States

Topics: Animals; Antigens; Cell Line; Cell Transformation, Neoplastic; Chromosomes; Clone Cells; Complement Fixation Tests; Cricetinae; Culture Techniques; Fibroblasts; Hybridization, Genetic; Kidney; Phenotype; Polyomavirus; Thioguanine

Topics: Animals; Antigens; Avian Leukosis Virus; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Chick Embryo; Clone Cells; Complement Fixation Tests; Cricetinae; Culture Media; Fibroblasts; Helper Viruses; Kidney; Phenotype; Polyomavirus; Polyploidy; Thioguanine; Virus Replication

A selective method was devised for the isolation of "revertants" from polyoma-transformed sublines derived from BHK21/13 Syrian hamster fibroblasts. A hybrid, polyploid subline was obtained by growing together, in mixed culture in the presence of aminopterin, two variant BHK21/13 sublines lacking either inosinic acid pyrophosphorylase or thymidine kinase. Whereas these variant sublines were resistant to 6-thioguanine or to 5-bromodeoxyuridine, the hybrid had regained sensitivity to both analogues. By plating a polyoma-transformed subline derived from this hybrid in the presence of 6-thioguanine, resistant clones were obtained with a frequency of about 10(-4). All of these surviving clones had a reduced chromosome complement and some of them had regained a normal phenotype.

Topics: Aminopterin; Animals; Bromodeoxyuridine; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes; Cricetinae; Culture Techniques; Fibroblasts; Glycine; Hybridization, Genetic; Hypoxanthines; Karyotyping; Kidney; Polyomavirus; Thioguanine; Thymidine