High frequency in vivo loss of heterozygosity is primarily a consequence of mitotic recombination.

We have used the adenine phosphoribosyltransferase gene (APRT; 16q24) to investigate the mechanisms of loss of heterozygosity (LOH) in normal human somatic cells in vivo. APRT-deficient (APRT-/-, APRT-/0) T lymphocytes from the peripheral blood of four obligate APRT heterozygotes (APRT+/-) with characterized germ-line mutations were selected in medium containing 100 microM 2,6-diaminopurine. A total of 80 2,6-diaminopurine-resistant T-cell clones from 2 of the heterozygotes were analyzed for this study. The presence or absence of LOH of proximal linked microsatellite repeat markers was used to divide the clones into two groups: (a) those in which LOH was likely due to localized changes in APRT (e.g., point mutations); and (b) those with LOH at additional loci. A total of 61 clones (76%) exhibited LOH of linked microsatellite repeat markers at different locations on 16q, which extended from the smallest measured region (<5.5 cM) to the entire 16q arm. The remaining 19 clones (24%) had point mutations in APRT or other relatively minor alterations. Ten clones with LOH encompassing different regions of 16q were examined by conventional cytogenetics and by fluorescence in situ hybridization using an APRT cosmid probe. All clones exhibited a normal diploid karyotype, and nine exhibited two copies of APRT. The one clone that was hemizygous for APRT had the smallest observed region of LOH in clones from that individual. These results indicate that mitotic recombination and, to a much lesser extent, deletion may be the primary mechanisms for the relatively high frequency of in vivo LOH observed in normal human T cells. Because LOH leads to the expression of recessive tumor suppressor genes in many cancers, these data have significant implications for the role of LOH in the early stages of tumor development, especially in breast cancer.

Analysis of human HPRT deletion mutations with X-linked probes and pulsed field gel electrophoresis.

Because the human hprt gene is used in numerous mutation studies, it is important to fully characterize this gene. Therefore, our laboratory has undertaken to map the region around the hprt gene at band q26 of the human X chromosome. Utilizing hprt mutant T-cell clones isolated using the hprt clonal assay, which have deletions of all or part of the hprt gene, we have ordered 5 anonymous probes previously known to map in Xq26. Results suggest that this region includes between 460 kb and 18 Mb of DNA, which is at least 10 times the size of the hprt gene itself (43 kb). Pulsed field gel analysis of the region is underway to determine the exact distances between each of the anonymous probes and hprt and to determine deletion sizes in the mutant T-cell clones.

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed.

Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes.

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.

Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.

A role for HEM2 in cadmium tolerance.

A Candida glabrata cadmium-sensitive mutant partially defective in glutathione production and exhibiting a complete absence of phytochelatins was used to clone a gene required for Cd tolerance. Transformation of the Cd-sensitive mutant with a genomic library from the wild-type C. glabrata led to the cloning of a gene that restored Cd tolerance and formation of Cd-glutathione and Cd-phytochelatin complexes. The cloned gene showed high levels of nucleic acid and protein sequence homology to the HEM2 genes, encoding porphobilinogen synthases, from several sources. It was shown that the C, glabrata Cd-sensitive mutant indeed exhibited a significant reduction in porphobilinogen synthase levels. The cloned C. glabrata gene complemented a hem2 mutant of Saccharomyces cerevisiae and restored porphobilinogen synthase activity in the mutant. The Cd sensitive mutant predictably showed decreased levels of sulfite reductase that requires siroheme, a metabolite produced in the heme biosynthetic pathway. The addition of cysteine, but not methionine, increased glutathione levels and Cd tolerance of both the wild-type and the mutant strain. However, addition of hemin chloride and methionine together restored Cd tolerance indicating that heme was required for transsulfuration of homocysteine to cysteine.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes. III. Longitudinal study of hprt gene structural alterations and T-cell clonal origins.

The hprt clonal assay detects mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes. Analysis of 94 wild-type and 326 hprt mutant clones from 3 normal males was performed using Southern blotting with hprt and T-cell receptor (TCR) gene probes. Gross structural alterations of the hprt gene occurred in approximately 14% of the in vivo derived mutants. Breakpoints were randomly distributed across the gene with one possible mutational "hot spot" observed. Most hprt mutants were independent as judged by TCR gene rearrangement patterns indicating that the measured hprt mutant frequency is a good measure of the actual hprt mutation frequency. However, sibling mutants (generally doublets and triplets except for one nonamer) were detected. Information on the timing in vivo of the hprt mutational events and the persistence in vivo of sibling mutants was also obtained.

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by gamma-irradiation.

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of gamma-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR beta and gamma cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (10/18) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5' and 3' regions of the hprt gene (4/18 and 4/18, respectively). These results demonstrate that gamma-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

Pulsed field analysis of hprt T-cell large deletions: telomeric region breakpoint spectrum.

In order to determine a large deletion breakpoint spectrum, 25 independent hprt T-lymphocyte mutants with deletions extending from hprt into the telomeric or centromeric flanking chromosomal region were analyzed by pulsed field gel electrophoresis (PFGE). PFGE was used to determine deletion sizes which allowed localization of breakpoints external to hprt to specific chromosomal positions in mutants containing an intra-hprt breakpoint. A breakpoint spectrum based on 19 large deletion mutants is reported for the Xq26 chromosomal region telomeric to hprt. A potential cluster of breakpoints (4/19) was observed approximately 60 kb from hprt. In addition, maximum recoverable deletion size was at least 3.5 Mb. Three of the 25 mutants analyzed appeared to be complex deletion events.

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes.

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.

The effect of T-lymphocyte 'clonality' on the calculated hprt mutation frequency occurring in vivo in humans.

The frequency of 6-thioguanine resistant (TGr) mutant T-lymphocytes arising in vivo in humans can be quantified with a cell cloning assay. However, the in vivo proliferation of T-lymphocytes that may include TGr mutant cells can distort the relationship between mutation events and the resulting frequency of mutant cells. The T-cell receptor (TCR) gene rearrangement pattern of T-cell colonies can be used as an independent measure of clonality. Analysis of T-cell 'clonality' in 413 wild type and 1736 TGr mutant isolates from 58 individuals shows that mutant clonality is a frequent occurrence (35/58 individuals = 60.3%). However, a major effect on the mutant frequency corrected for clonality (the calculated 'mutation frequency') was found only in nine samples all of which had mutant frequencies greater than 40 x 10(-6).

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells.

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Role of CdS quantum crystallites in cadmium resistance in Candida glabrata.

Glutathione-related peptides (gamma-Glu-Cys)nGly, trivially known as phytochelatins (PCs), sequester Cd(II) and other heavy metals in all plants and some yeasts. However, the metal resistance levels may depend on factors such as the PC concentrations, their chain length and ability to incorporate labile sulfide. We show here that a highly Cd(II)-resistant mutant of yeast Candida glabrata exhibited Cd(II)-dependent formation of extremely high levels of PC-coated CdS quantum crystallites. The CdS crystallites were formed in the cytosol but finally accumulated in the vacuoles. Cd(II)-stimulated sulfide production required sulfate and was inhibited by both cysteine and methionine. GSH synthesis inhibition sensitized the resistant strain to Cd(II) indicating that GSH still provided primary defense against the metal ion.

Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.

Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability.

Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins.

Optical spectroscopy and reverse-phase HPLC were used to investigate the binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (gammaGlu-Cys)2Gly, (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly. Glutathione-mediated transfer of Hg(II) into phytochelatins and the transfer of the metal ion from one phytochelatin to another was also studied using reverse-phase HPLC. The saturation of Hg(II)-induced bands in the UV/visible and CD spectra of (gammaGlu-Cys)2Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecule. The separation of apo-(gammaGlu-Cys)2Gly from its Hg(II) derivative on a C18 reverse-phase column also indicated the same metal-binding stoichiometry. The UV/visible spectra of both (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280-290 mm. Two distinct Hg(II)-binding species, occurring at metal-binding stoichiometries of around 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (gammaGlu-Cys)3Gly. These species exhibited specific spectral features in the charge-transfer region and were separable by HPLC. Similarly, two main Hg(II)-binding species of (gammaGlu-Cys)4Gly were observed by UV/visible and CD spectroscopy at metal-binding stoichiometries of around 1.25 and 2.5 respectively. Only a single peak of Hg(II)-(gammaGlu-Cys)4Gly complexes was resolved under the conditions used for HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins were similar at pH 2.0 and at pH 7.4, indicating that pH did not influence the final Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer of the metal ion from shorter- to longer-chain phytochelatins. The strength of Hg(II) binding to glutathione and phytochelatins followed the order: gammaGlu-Cys-Gly<(gammaGlu-Cys)2Gly<(gammaGlu-Cy s)3Gly<(gamma Glu-Cys)4Gly.

Analysis of T cell receptor beta and gamma genes from peripheral blood, regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients.

A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4+CD8-, although some were CD4-CD8+. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) beta and gamma gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. Probing HindIII-digested DNA with TCR beta and TCR gamma probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing of BamHI-digested DNA with TCR beta and TCR gamma probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4+ T cell populations in each of several separate immune compartments in these patients.

Molecular analysis of in vivo hprt mutations in human T lymphocytes. V. Effects of total body irradiation secondary to radioimmunoglobulin therapy (RIT)

The hprt (hypoxanthine guanine phosphoribosyltransferase) T cell cloning assay was used to detect in vivo mutations in T lymphocytes of individuals receiving radioimmunoglobulin therapy (RIT). A total of 28 patients receiving 131I and/or 90Y-labeled antiferritin antibodies was studied. Mutant frequencies for patients were clearly much higher than for historic non-treated controls (median 68.0 X 10(-6) for patients versus a median of 6.8 X 10(-6) for 115 controls). There was a good correlation of mutant frequency with initial activity of RIT (rlinear = 0.68, rquadratic = 0.76; P less than 0.05) although the correlation of mutant frequency with total activity after several rounds of treatment was poor (R = 0.18). Molecular studies of the hprt mutants demonstrated that a much higher proportion of mutations occurring in RIT treated patients had gross structural alterations of the hprt gene (33%) than did mutations occurring in controls (15%). There was a good correlation (r = 0.72) of mutants with gross alterations and total RIT activity. T cell receptor gene studies demonstrated that most of the mutants (92%) represented independent in vivo mutations, which is similar to previous findings with background mutations in non-irradiated individuals. These studies demonstrate the usefulness of the hprt T cell cloning assay for studies of in vivo human somatic cell gene mutations resulting from ionizing radiation.

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures.

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (I5: +1, delta G, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag67/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (I5:2-4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2-6. In addition to defining a new Lesch-Nyhan mutation (hprtHenryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.