6-thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity.

Cell populations resistant to high doses (30 microM) of 6-thioguanine (6-TG, 6-TG(r) cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSalpha, is resistant to low doses of 6-TG. The level of activity of hypoxanthine-guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6-TG or its derivatives into non-toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6-TG in the 2 cell types. Net uptake of 6-TG ceased after 100-sec incubation in the 6-TG(r) cells, while it appeared to continue throughout the 10-min incubation in the wild-type cells. As a consequence, after 10-min incubation, the total amount of 6-TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6-TG(r) cells.     

Induction of hypoxanthine phosphoribosyltransferase deficiency in human U-937 cells

The aim of the study was to establish enzyme-deficient mutants of the human permanent cell line U-937. Following chemical mutagenesis with the use of ethyl methanesulfonate, this cell line was chronically exposed to increasing concentrations of the toxic hypoxanthine analogue 6-thioguanine. Cells surviving hypoxanthine-aminopterin-thymidine selective media were separated by glass adherence with the use of 12-O-tetradecanoyl-phorbol-13-acetate. Three mutant clones were established, which have remained hypoxanthine phosphoribosyltransferase (HPRT) deficient for a period of 7 months, as shown by indirect measurements with the use of autoradiography and scintillation counting of cells exposed to [3H]hypoxanthine. Since the phenotypic properties and growth behavior of U-937 cells have remained unaltered after the induced mutation, a highly restricted chromosomal segment coding for HPRT seems to have been mutated.     

In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclicaromatic hydrocarbon with potent mutagenic and carcinogenicactivity. In order to acquire an understanding of the mutagenicpathways of CPP, we studied mutations induced by this chemicalin human cells. Four independent cultures of a human cell lineexpressing cytochrome P450 CYP1A1 (cell line MCL-5) were treatedwith CPP, and mutants at the hypoxanthine phosphoribosyltransferase(HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance.The kinds and positions of the mutations were analyzed usingthe combination of high-fidelity polymerase chain reaction (hifi-PCR)and denaturing gradient gel electrophoresis (DGGE). The thirdexon of the HPRT gene was amplified from the 6TG-resistant cellsusing the hifi-PCR and the amplified fragment was subsequentlyanalyzed by DGGE to separate mutant sequences from the wild-typesequence. Mutant bands were excised from the gel, amplifiedusing PCR and sequenced. Sixteen different mutations were identifiedand consisted mostly of the G to T and A to T transversions.Other mutations identified included G to A and A to G transitions,a G to C transversion, and a single G deletion. Of these mutations,six occurred within a run of six guanines. The predominanceof transversions involving a guanine or an adenine observedwith CPP is similar to the data previously reported for theracemic mixture of benzo[a]pyrene (B[a]P), suggesting that themechanisms of mutation induced by CPP may be similar to thoseinduced by B[a]P.     

Hypoxanthine:guanine phosphoribosyltransferase activity in xenografts of human osteosarcoma

It has recently been reported that human osteosarcomas may lack the purine salvage pathway enzyme, hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8). We have established a quantitative assay for measurement of this enzyme in human osteosarcoma xenografts with analysis of products by thin-layer chromatography. Nucleotidase or phosphatase activity was readily detected and could be abolished by preheating cytosol at 60 degrees C for 10 min and performing the assay at pH 10. Alternatively, the use of 25 mM NaF at pH 7.4 also inhibited this activity. The pH optimum for this enzyme in red blood cell sonicates and tumor cytosols was pH 10. All six human osteosarcoma xenografts contained hypoxanthine:guanine phosphoribosyltransferase activity ranging from 0.97 to 4.06 nmol/min/mg of protein at pH 7.4. Control human red blood cell sonicates demonstrated activity of 0.83 nmol/min/mg of protein. These data demonstrate that human osteosarcoma xenografts contain substantial activities of this purine salvage pathway enzyme.     

Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (hprt) gene of healthy people

Understanding the significance of somatic mutations requiresknowledge of the mutations that occur in vivo in healthy people.The molecular characterization of mutations in the hypoxanthinephosphoribosyltransferase (hprt) gene in 217 independent T-lymphocytemutants from 172 donors, including smoking and non-smoking malesand females, reveals a broad spectrum of in vivo somatic mutationoccurring in a population of healthy people. Identificationof the DNA alteration in individual mutant clones was accomplishedusing either one or a combination of multiplex polymerase chainreaction analysis of genomic DNA, sequencing of cDNA, and genomicDNA sequencing. The total spectrum consists of 59% (128/217)base substitutions: 126 simple and two tandem CC>TT basesubstitutions; 39% (85/217) deletion/insertion type mutations:30 frameshifts, 26 small (3–200 basepairs) and 27 largedeletions, and two duplications; and the remaining 2% (4/217)complex mutations involving the deletion of one to 11 basepairswhich are replaced by 1 to 10 basepairs. No significant differencewas detected between the base substitution spectra for the smokersand the non-smokers. Analysis of the number of mutations occurringat any one base position led to the identification of threehotspots for mutations at basepairs 197, 508 and 617, in thehprt gene coding region. Spontaneous deamination of CpG maybe implicated in the creation of basepair 508 as a hotspot sinceall mutations detected are C>T transitions resulting in thenonsense mutation, TAG. At basepairs 197 and 617 both G>Ttransversions and G>A transitions were found indicating thatat least two mechanisms were involved in creating mutationsat these positions. Comparison of the mutation spectra fromtwo populations can provide insight into the origin of the mutations.This study provides an excellent base for comparison of mutationspectra in other human populations.     

Molecular analysis of spontaneous hypoxanthine phosphoribosyltransferase mutations in thioguanine-resistant HL-60 human leukemia cells

We have measured the forward mutation rate at the hypoxanthine phosphoribosyltransferase (HPRT) gene of the human promyelocytic leukemia cell line HL-60 and have determined the molecular spectrum of spontaneous HPRT mutations in 45 independent 6-thioguanine-resistant HL-60 sublines. Four fluctuation tests using a total of 132 replicate HL-60 cultures revealed a mean forward mutation rate of HL-60 cells to thioguanine resistance of 1.7-6 x 10(-7)/cell/generation. Blot hybridization analysis of the X-linked HPRT gene using a human HPRT complementary DNA probe revealed abnormalities in HPRT gene structure and/or HPRT mRNA expression in 24 of 45 (53%) independent thioguanine-resistant HL-60 sublines. Six different classes of mutation were identified. The most prevalent (47%; 21 of 45 mutations) consists of mutations that are not detected by blot hybridization analyses and that do not disrupt HPRT mRNA production. These results suggest that a comparatively low forward mutation rate may be found in malignant human cells that exhibit both karyotypic and molecular evidence of genomic instability and that several different molecular classes of mutation may contribute to thioguanine resistance in HL-60, and perhaps in other, malignant human cells. The forward mutation assay system we have developed using the X-linked HPRT gene of HL-60 cells may be useful for analyses of the mutagenic potential and molecular spectrum of mutations produced by chemotherapeutic agents, suspected human mutagens and carcinogens, and phagocyte respiratory burst oxidants in human cells.     

Biomarkers of styrene exposure in lamination workers: levels of 06-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes

Occupational exposure to styrene was studied in nine workersof a hand lamination plant in Bohemia. Personal dosimeters wereused to monitor the styrene workplace exposure, and the levelsof styrene in blood and mandelic acid in urine were measured.Blood samples were taken at four occasions during a 7 monthperiod to determine styrene-specific 0⁶-guanine DNA adductsin lymphocytes and granulocytes, DNA strand breaks and hypoxanthineguanine phosphoribosyltransferase (HPRT) mutant frequency inT-lymphocytes. Seven administrative employees in the same factory(factory controls) and eight persons in a research laboratory(laboratory controls) were used as referents. DNA adduct levelsdetermined by the ³²P-postlabelling method in lymphocytes oflamina-tors were remarkably constant and significantly higher(P < 0.0001) than in factory controls at all four samplingtimes. HPRT mutant frequencies (MF) measured by the T-cell cloningassay were higher in the laminators (17.5 x10^–6, groupmean) than in the factory controls (15.7x10^–6, group mean)at three of the four sampling times, but the differences werenot statistically significant. However, a statistically significant(P = 0.021) difference between MF in the laminators (18.0 x10^–6,group mean) and laboratory controls (11.8 xl0^–6, groupmean) was observed at sampling time 4 (the only sampling timewhen this latter group was studied). This result indicates thatstyrene exposure may induce gene mutation in T-cells in vivo.DNA strand breaks were studied by the ‘Comet assay’at the fourth sampling time. The laminators were found to havesignificantly higher levels of DNA strand breaks than the factorycontrols (P = 0.032 for tail length, TL; P = 0.007 for percentageof DNA in tail, T%; and P = 0.020 for tail moment, TM). A statisticallysignificant correlation was also found between the levels oflymphocyte DNA adducts and all three DNA strand break parameters(TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary,no significant correlations were found between DNA adduct levelsand the HPRT mutant frequencies or between the mutant frequenciesand DNA strand breaks. Taken together, these results add furthersupport to the genotoxic and possibly mutagenic effects of styreneexposure in vivo. However, no simple quantitative relationshipseems to exist between the levels of styrene-induced DNA damageand frequency of HPRT mutation in T-lymphocytes.     

Spectrum of point mutations in the coding region of the hypoxanthine- guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes in vivo

The hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in 6- thioguanine (TG) resistant T-lymphocytes is a useful target for the study of somatic in vivo mutagenesis, since it provides information about a broad spectrum of mutation. Mutations in the hprt coding region were studied in 124 TG-resistant T-cell clones from 38 healthy, non- smoking male donors from a previously studied population of bus maintenance workers, fine-mechanics and laboratory personnel. Their mean age was 43 years (range 23-64) and their hprt mutant frequency was 9.3 +/- 5.2 x 10(-6) (mean +/- SD, range 1.4-22.6 x 10(-6)). Sequence analysis of hprt cDNA identified 115 unique mutations; 76% were simple base substitutions, 10% were +/-1 bp frameshifts, and 10% were small deletions within exons (3-52 bp). In addition, two tandem base substitutions and one complex mutation were observed. Simple base substitutions were observed at 55 (20%) of 281 sites known to be mutable in the hprt coding sequence. The distribution of these mutations was significantly different than would be expected based upon a Poisson distribution (P < 0.0001), suggesting the existence of 'hotspots'. All of the 87 simple base substitutions occurred at known mutable sites, but eight were substitutions of a kind that have not previously been reported at these sites. The most frequently mutated sites were cDNA positions 197 and 146, with six and five independent mutations respectively. Four mutations were observed at position 131, and three each at positions 143, 208, 508 and 617. Transitions (52%) were slightly more frequent than tranversions (48%), and mutations at GC base pairs (56%) more common than mutations at AT base pairs (44%). GC > AT was the most common type of base pair substitution (37%). The majority of the mutations at GC base pairs (78%) occurred at sites with G in the non-transcribed strand. All but one of eight mutations at CpG- sites were of the kind expected from deamination of methylated cytosine. Deletion of a single base pair (-1 frameshift) was three times more frequent than insertion of a single bp (+1 frameshift). Almost half (6/13) of the small (3-52 bp) deletions within the coding sequence clustered in the 5' end of exon 2. Short repeats and other sequence motifs that have been associated with replication error were found in the flanking regions of most of the frameshifts and small deletions. However, several differences in the local sequence context between +/-1 frameshift and deletion mutations were also noticed. The present results identify positions 197, 146 and possibly 131 as hotspots for base substitution mutations, and confirm previously reported hotspots at positions 197, 508 and 617. In addition, the earlier notion of a deletion hotspot in the 5'end of exon 2 was confirmed. The observations of these mutational cluster regions in different human populations suggest that they are due to endogeneous mechanisms of mutagenesis, or to ubiquitous environmental influences. The emerging background spectrum of somatic in vivo mutation in the human hprt gene provides a useful basis for comparisons with radiation or chemically induced mutational spectra, as well as with gene mutations in human tumors.      

Endogenous gene systems for the study of mutational specificity in mammalian cells

Determination of the DNA sequence alterations produced by various mutagens is a prerequisite for understanding mechanisms of mutagenesis. With recent technical advances that permit rapid isolation of mutant alleles, the mammalian genome has become accessible to this type of analysis. Here we discuss the growing data base on mutational specificity in mammalian cells, with an emphasis on the information obtained from studies of two endogeneous genetic loci, aprt and hprt.     

In vitro schedule-dependent interaction between paclitaxel and cisplatin in human carcinoma cell lines

 The schedule-dependent interaction of paclitaxel and cisplatin was studied in four human carcinoma cell lines: non-small cell lung cancer, A549; breast cancer, MCF7; ovarian cancer, PA1; and colon cancer, WiDr cells. The cells were exposed simultaneously to the drugs for 24 h and sequentially to paclitaxel first for 24 h followed by cisplatin for 24 h, or vice versa, and then incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was then determined by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. On simultaneous exposure to paclitaxel and cisplatin, additive and sub-additive (slight antagonistic) effects were observed in A549, MCF7, and PA1 cells, while sub-additive and protective (antagonistic) effects were observed in WiDr cells. On sequential exposure to paclitaxel first, followed by cisplatin, additive effects were observed in all cell lines. On sequential exposure to cisplatin first, followed by paclitaxel, additive effects were observed in PA1 cells, while additive, sub-additive, and protective effects were observed in A549, MCF7, and WiDr cells. These findings suggest that the interaction of paclitaxel and cisplatin is schedule- and cell line-dependent. The optimal schedule of this combination may be paclitaxel first followed by cisplatin. Received: 15 November 1994/Accepted: 21 June 1995     

Hypoxanthine:guanine phosphoribosyltransferase activity in primary human osteosarcomas. A rationale for therapy with methotrexate-thymidine rescue?

Hypoxanthine:guanine phosphoribosyltransferase (HPRT) activity was measured in 14 human osteosarcomas to test whether a subset of these tumors was deficient in the purine salvage pathway enzyme and thus provide a rationale for therapy with methotrexate-thymidine rescue. All tumors contained HPRT activity within the range previously reported for xenografts of human osteosarcoma. Three patients received methotrexate (3.375 g/m2/24 hours) as a 72-hour continuous infusion with thymidine rescue (2.0 g/m2/24 hours) beginning 24 hours after the start of the methotrexate infusion. The methotrexate-thymidine infusion was well tolerated by all patients with no significant toxicity; however, there were no responses. We conclude that osteosarcomas are not deficient in HPRT activity. Therefore, the previously reported rationale for therapy of osteosarcoma with methotrexate-thymidine based on lack of activity of this enzyme is not valid. This combination, although well tolerated, is inconvenient and requires prolonged hospitalization. Therefore, without a valid rationale it cannot be recommended for therapy of patients with osteosarcoma.     

Sequence specificity of mutation induced by the anti-tumor drug cisplatin in the CHO aprt gene

Cis-diammine dichloroplatinum (cisplatin) is an effective anticancerdrug which forms adducts with DNA, in both bacterial and mammaliancells. It is suspected of producing tumors as well. To determinethe molecular nature of geneti alterations induced by cisplatin,we cloned and sequenced cisplatin-induced mutants in the adeninephosphoribosyl-transferase (aprt) gene of Cinese hamster ovary(CHO) cells. Mutation by cisplatin appears to be targeted asthe sites of mutation are consistent with the known bindingspecificity of cisplatin. Many mutations occur at or proximalto the sequence 5'-AGG-3' and 5'-GAG-3' and include transversions,transitions, frameshifts and short deletions and duplications.Several double changes were also observed. No major rearrangementswere recovered in our collection. At several locations, a numberof mutants were found to be clustered within a small targetregion, but unlike traditional hotspots, tese represent diversechanges occurring in a localized region of a few base pairs.     

Variables and specificity of in vitro lymphocyte-mediated cytotoxicity in human melanoma.

In vitro cell-mediated cytotoxicity (CMC) assays have been carried out in human melanoma system with blood effector lymphocytes on [3H]proline-labeled target cells in a 48-hr microcytotoxicity technique. Three lymphocyte purification procedures (Ficoll:Hypaque gradient, plasma gel sedimentation followed by nylon column incubation, and plasma gel sedimentation followed by separation with nylon powder and glass beads) are compared in parallel experiments for characteristic effector cell composition and cytotoxic potential against target cells of dissimilar histology. The cytotoxicity is defined by the loss of target cell 3H cpm as measured by residual target cell 3H cpm in individual microwell following incubation with lymphocytes. Target cell 3H cpm loss by test lymphocytes is compared with target cell 3H cpm loss by several age and sex matched control lymphocytes (from normal donors and unrelated cancer patients); further comparison between the various control lymphocytes is also made in each assay. As control for target cells, autologous fibroblasts and homologous tumor cells of dissimilar histology are always included in each assay. Specific cytotoxicity is defined as statistically significant and selective destruction of only melanoma cells by the test lymphocytes as compared to the control lymphocytes. Significant but nonselective destruction of 2 or more target cells of unrelated histology is regarded as nonspecific cytotoxicity, while no destruction of any target cells signifies no cytotoxicity. The Ficoll:Hypaque preparations consistently exhibit the highest nonlymphocytic cell contamination (8 to 16%); the nonlymphocytic cells are, almost exclusively, monocytes. They also produce relatively high percentage of thymus independent (B) cells (8 to 15%). The ultimate cell composition of the 2 plasma gel-nylon preparations is essentially identical. In either plasma gel-nylon preparations, the nonlymphocytic contamination is minimal (0 to 4%) and thymus-dependent (T) cell percentage is considerably higher (92 to 99%) with none or few B cells.     

DNA sequence specificity of doxorubicin-induced mutational damage in uvrB- Escherichia coli.

In the absence of excision repair, doxorubicin caused a striking (41-fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac repressor gene (lacI) of Escherichia coli. In contrast, there was only a 2-fold increase in the frequency of small deletions despite a 3-fold increase in overall mutation frequency. The 5'-endpoints of doxorubicin-induced lacO and lacI/lacO deletions occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent to 5'-dGdC or 5'-dCdG doublets (34%). Ninety % (27 of 30) of the doxorubicin-induced deletions involving the region of the lacO palindrome had 3'-endpoints within the palindrome sequence as compared with 40% (4 of 10) spontaneous deletions in an untreated set. Doxorubicin-induced single base substitutions were highly focused at one site (4 of 6) in the i-d region of lacI, in contrast to the spontaneous distribution of point mutations, where 16 mutants were recovered at 12 different sites. An increased frequency (3-fold) of highly focused base substitutions was also observed at 2 sites in the lac operator region (at lacO +6, which is a transition "hotspot" in the spontaneous spectra of both wild type and uvrB- organisms and at the adjacent +5 site). Notably, the frequency of 1- and 2-base frameshifts did not increase in the doxorubicin-induced spectrum, relative to the spontaneous mutation spectrum. These in vivo observations in E. coli suggest that in the absence of excision repair, doxorubicin causes highly focused deletions and base substitutions. These mutations occur adjacent to DNA sequences identified in previous in vitro studies as preferential sites of doxorubicin binding.     

Circumvention of 5-fluorouracil resistance in human stomach cancer cells by uracil phosphoribosyltransferase gene transduction.

A human stomach cancer cell line with acquired resistance to 5-fluorouracil (5-FU), NUGC-3/5FU/ L, has been found to possess reduced ability to convert 5-FU into active metabolites. We attempted in vitro gene therapy for this 5-FU-resistant cell line. NUGC-3 and NUGC-3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA-UPRT, and changes in their 5-FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC-3 and NUGC-3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5-FU nucleotide level in the acid-insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC-3 cells on infection with AdCA-UPRT, and in NUGC-3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth-inhibition concentration (IC50) was 12.7 micromol/liter for NUGC-3 and much higher than 100 micromol/liter for NUGC-3/5FU/L, indicating over 8-fold resistance. NUGC-3/ SFU/L transfected with the UPRT gene showed very high sensitivity to 5-FU with an IC50 of 3.2 micromol/liter. The high resistance in this metabolic activation-deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5-FU-anabolizing enzyme.     

Synergism between dipyridamole and cisplatin in human ovarian carcinoma cells in vitro.

Dipyridamole (DPM), a nucleoside membrane transport inhibitor, enhanced the cytotoxicity of cisplatin (DDP) for human ovarian carcinoma 2008 cells by a factor of 4.7 +/- 0.4-fold (mean +/- SD) and for the 10-fold DDP-resistant 2008/C13*5.25 subline by a factor of 5.8 +/- 2.7-fold. This interaction was shown to be truly synergistic by isobologram and median effect analysis. DPM enhancement of DDP cytotoxicity was schedule dependent; it was greatest when cells were exposed to DPM continuously during and following a 1-h exposure to DDP and less pronounced when DPM exposure was limited to pretreatment or concurrent treatment only. DPM increased DDP uptake in a concentration-dependent manner as measured with both [195mPt]-DDP and the DDP analogue [3H]-cis-dichloro(ethylenediamine) platinum. Nitrobenzylthioinosine, another nucleoside membrane transport inhibitor, did not enhance DDP cytotoxicity or uptake at concentrations that produced equivalent degrees of inhibition of [3H]uridine uptake. DPM did not interact synergistically through an increase in cellular cyclic AMP levels. DPM did not increase trypan blue or propidium iodide uptake, or change cell size, indicating that it did not nonspecifically increase membrane permeability. We conclude that DPM interacts synergistically with DDP and that, while an increase in DDP uptake is one component of the mechanism of this interaction, there are additional components since maximal effect was observed only with prolonged DDP exposure.     

In vitro cultivation of human tumor tissues.

In order to develop cell substrates suitable for the isolation of human oncornaviruses, a large number of human tumors, mostly sarcomas, were sultured in vitro. All explants yielded primary outgrowth of cells. These were of various nature and morphology and exhibited different growth potentials. About 40% of the tumors yielded cell strains which were undistinguishable from diploid human embryonic cell strains. About 20% yielded 'difficult' diploid cultures; 35% yielded short-term cultures mostly composed of undefinable cells, rarely of clearly recognizable tumor cells. About 5% yielded cell strains or lines which were aberrant in morphology or karyotype, or both. The malignant nature of the cell strains obtained in our study is conjecturable. Although some of the cell cultures showed evidence of tumoral origin, none complied with currently employed in vitro criteria for malignancy such as lack of topoinhibition and high density growth.