Skin color and nutrient photolysis: an evolutionary hypothesis.

Human populations native to areas of intense sunlight tend to be heavily melanized. Previous explanations for this relationship have invoked only weak selective pressures. To test the hypothesis that dark pigmentation may protect against photolysis of crucial light-sensitive vitamins and metabolites by ultraviolet light, folate was used as a model. It was found that exposure of human plasma in vitro to simulated strong sunlight causes 30 to 50 percent loss of folate within 60 minutes. Furthermore, light-skinned patients exposed to ultraviolet light for dermatologic disorders have abnormally low serum folate concentrations, suggesting that photolysis may also occur in vivo. Deficiency of folate, which occurs in many marginally nourished populations, causes severe anemia, fetal wastage, frank infertility, and maternal mortality. Prevention of ultraviolet photolysis of folate and other light sensitive nutrients by dark skin may be sufficient explanation for the maintenance of this characteristic in human groups indigenous to regions of intense solar radiation.

The mechanism of 5-methyltetrahydrofolate transport by human erythrocytes.

The mechanism involved in 5-methyltetrahydrofolate uptake by human cells is poorly understood. To more clearly elucidate this physiologically important process, transport of the vitamin was studied in human erythrocytes. 5-methyltetrahydrofolate uptake was found to increase with reticulocytosis, but measurable incorporation occurred in erythrocyte suspensions depleted of reticulocytes, leukocytes, and platelets, indicating uptake by mature erythrocytes. Incubation of erythrocytes with increasing concentrations of [(14)C]5-methyltetrahydrofolate resulted in increasing uptake but decreasing percentage incorporation, consistent with saturation of a carrier system. Both influx and efflux phases of uptake were temperature dependent, with almost no transport at 4 degrees C. Uptake of [(14)C]5-methytetrahydrofolate was effectively inhibited by unlabeled 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but not by pteroylglutamic acid. Prior incubation with 5-formyltetrahydrofolate increased uptake of [(14)C]5-methyltetrahydrofolate, and extracellular 5-formyltetrahydrofolate enhanced efflux of [(14)C]5-methyltetrahydrofolate. Nearly total depletion of ATP increased uptake of [(14)C]5-methyltetrahydrofolate, but efflux was unchanged. Column chromatography of membrane-free hemolysate after incubation with [(14)C]5-methyltetrahydrofolate showed 95% of radioactivity corresponded to marker radioisotope, and no other peak was noted. Thus peripheral erythrocytes incorporate 5-methyltetrahydrofolate by a saturable, temperature-dependent, substrate-specific process which is influenced by counter-transport. This mechanism is qualitatively similar to the carrier-mediated transport of folate compounds previously described in other cell types. Therefore, human erythrocytes should be useful for detailed characterization of this membrane carrier system.

Folate deficiency increases genetic damage caused by alkylating agents and gamma-irradiation in Chinese hamster ovary cells.

The effect of folate deficiency on genetic damage caused by alkylating agents and gamma-irradiation was studied in Chinese hamster ovary (CHO) cells. Mutant frequencies of 6-thioguanine-resistant and diphtheria toxin-resistant cells were not significantly increased by incubation in low-folate medium. In contrast, folate deficiency increased the mutant frequencies of 6-thioguanine-resistant cells caused by N-ethyl-N-nitrosourea or ethyl methanesulfonate by about 3-fold. Folate deficiency was associated with a 70% increase of diphtheria toxin-resistant cells after exposure to ethyl methanesulfonate. Folate deficiency alone caused DNA strand breaks equivalent to 26 cGy, as monitored by alkaline filter elution. Following 400 cGy of gamma-irradiation, folate-deficient cells manifested strand breaks equivalent to a dose of 710 cGy. CHO cells in folate-containing medium repaired breaks within 3 h, while cells in low-folate medium had an increased break frequency (P = 0.02) at 3 h and were unable to fully repair radiation-induced damage even after 9 h. These studies indicate that folate deficiency acts synergistically with alkylating agents to increase somatic mutation and with gamma-irradiation to promote DNA strand breaks in CHO cells. Folate deficiency appears to potentiate the genetic damage caused by mutagens/carcinogens by limiting DNA repair.

Further characterization of the in vitro tumoricidal activity of staphylococcal protein A.

Treatment of plasma or serum from leukemic patients with solid phase staphylococcal Protein A induced leukemic blast cell lysis in vitro, but this effect was relatively independent of the amount of immunoglobulin G (IgG) removed. Samples with approximately equal cytotoxic activity contained markedly different IgG levels, while samples with similar IgG levels had a wide range of tumoricidal activity. Assays of plasma samples collected during a perfusion of one plasma volume through a Protein A-Sepharose column indicated that the duration of the procedure had a greater effect on cytotoxic activity than did the amount of IgG removed. Neither added leukemic nor normal IgG significantly improved blast cell viability in treated serum. Cytotoxic activity was not dialyzable and concentrated in the Mr less than 100,000 fraction of samples separated by filtration. Treated cytotoxic serum samples did not have important Clq binding activity. These results suggest that the in vitro tumoricidal activity of solid phase Protein A is probably due to a toxic substance added to serum during immunoadsorption rather than to its immunoadsorptive capacity.

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents.

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.

Effects of plasma treatment with purified protein A and Staphylococcus aureus Cowan I on spontaneous animal neoplasms.

Eleven dogs with spontaneous neoplasms were intensively treated with an immunoadsorption system consisting of a continuous flow centrifuge, Protein A-Sepharose columns, and a semi-automatic elution system. Despite consistent and substantial lowering of immunoglobulin G levels, tumor regression was noted in only one of 11 dogs. In contrast, infusion of small volumes of plasma after incubation with heat and formalin-treated Staphylococcus aureus Cowan I resulted in a tumoricidal response in five of six animals. These results suggest that tumor necrosis is probably not induced by Protein A-mediated removal of humoral "blocking" factors.

Factors influencing mutation at the hprt locus in T-lymphocytes: women treated for breast cancer.

Forty-nine women with breast cancer were enrolled in a prospective, longitudinal study of the genetic damage caused by treatment. Assays of mutant frequency at the hprt locus in peripheral blood lymphocytes were performed at approximately 6-month intervals for 2 years. Treatment consisted of surgery alone or additional tamoxifen, radiotherapy, or chemotherapy in various combinations. At 6 months, there was an elevation of mean mutant frequency compared to initial values (P = 0.004) which persisted for as many as 2 years. A significant elevation at 6 months occurred only in the group of women who received combination chemotherapy (P = 0.005). Within this group, 5 of 15 patients had striking elevations of mutant frequency following chemotherapy (greater than 3 SD). Three of these 5 women had serum folate levels in the deficient range, while only one of 9 patients with lesser responses to chemotherapy were folate deficient. The change in mutant frequency after chemotherapy was inversely related to serum folate levels (P = 0.05) and to the number of years of smoking cigarettes (P = 0.01). We conclude that of the various modalities used to treat breast cancer, only chemotherapy was accompanied by a high risk of somatic mutation. A subset of patients manifested substantial increases in mutant frequency, often in association with low serum folate levels.

Effects of folate deficiency on the metastatic potential of murine melanoma cells.

Experiments were designed to measure the effect of folic acid deficiency on a major determinant of cancer lethality, the propensity to form metastases. Murine B16 melanoma cells (F10 strain) were grown in folate-deficient and -supplemented media. After 3 days, cells in the deficient medium had restricted proliferative capacity, low folate levels by bioassay, increased cell volume, abnormal deoxyuridine suppression tests, accumulation of cells in S phase by flow cytometry, and increased numbers of DNA strand breaks. These folate-deficient cells consistently initiated more pulmonary metastases than control cells when injected into host mice. Cell size did not appear to be a major factor in pulmonary metastasis formation. In vitro growth rates and cloning efficiencies were comparable for cells in both types of medium as was subcutaneous growth of tumors. We conclude that folate deficiency increases the metastatic potential of cultured melanoma cells.

Phase I trial of intraperitoneal recombinant interleukin-2/lymphokine-activated killer cells in patients with ovarian cancer.

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and 27. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.

Oral anticoagulation thresholds.

BACKGROUND: Monitoring patients on oral anticoagulation is essential to prevent hemorrhage and recurrent thrombosis. We studied tissue factor-induced whole-blood coagulation in patients on warfarin therapy with similar international normalized ratios (INRs). METHODS AND RESULTS: Contact pathway-suppressed whole-blood coagulation initiated with tissue factor was studied in 8 male subjects (group W) and in 1 individual multiple times (subject A). Coagulation profiles for group W showed that subjects with similar INRs had widely varying clot times (6.2 to 23 minutes) and thrombin-antithrombin III (TAT) profiles with rates of 25 to 40 nmol. L(-1). min(-1) and maximum levels varying from 192 to 349 nmol/L. The normal control group exhibited clot times of 5.7+/-0.3 minutes and TAT rates of 57+/-13 nmol. L(-1). min(-1), reaching maximum levels of 742+/-91 nmol/L. Subject A, who was stably anticoagulated at an INR of 2.1+/-0.4 for 6 months, had widely ranging profiles with clot times of 9.0 to 22.7 minutes, TAT maximums varying from 141 to 345 nmol/L, and TAT formation rates of 10 to 57 nmol. L(-1). min(-1). INR did not correlate with TAT formation. Platelet activation was decreased by anticoagulants but also displayed variability. Fibrinopeptide A generation showed threshold variability independent of the INR. Factor VIII levels were increased (P=0.03) in group W (204+/-34.4%) compared with normal control subjects (149.4+/-37.4%). A significant correlation was identified between increasing factor VIII levels and years on warfarin therapy (r=0.78, P=0.01), suggesting a possible factor VIII compensatory mechanism. CONCLUSIONS: These results suggest that control of anticoagulation in patients to a set INR therapeutic range may be less secure than anticipated. Patients with similar INRs show significant individual variability in their tissue factor coagulation response, suggesting different risks to anticoagulation when confronted with underlying vascular anomalies.

Randomized study of nandrolone therapy for anemias due to bone marrow failure.

A prospective, randomized, double-blind study was designed to determine the effectiveness and toxicity of nandrolone phenpropionate in the treatment of anemias due to bone marrow failure. Twenty-four patients were initially entered; 21 now may be evaluated: seven with aplastic anemia, six with myelofibrosis, and eight with refractory anemia. Six patients improved, but only three were taking nandrolone, the other three placebo. Response did not correlate with type of anemia. No serious drug toxicity was noted. One patient with myelofibrosis improved dramatically with placebo therapy alone, no longer requiring frequent transfusions because of a hemoglobin level increase from 5.4 to 15.8 gm/100 ml. We conclude that no substantial improvement of anemia due to marrow failure can be ascribed to nandrolone as given, and that clinical trials in these conditions should be controlled to exclude spontaneous remissions as a cause of apparent improvement.

Folate metabolism and chromosomal stability in the fragile X syndrome.

Folate metabolism and the effects of folic acid on chromosome stability were studied in four related patients with the fragile X syndrome. In three adults, uptake and subsequent utilization of folate compounds for conversion of deoxyuridylate to thymidylate by marrow cells and stimulated lymphocytes, and the affinity and maximal transport velocity of erythrocyte membrane carriers, were normal. Numbers of sister chromatid exchanges and double-stranded DNA breaks were comparable in cells from patients and control subjects, but both were increased after incubation in folate-deficient media. In vitro expression of the fragile site was strikingly reduced by oral folate therapy. It is concluded that the folate-sensitive chromosomal defect in this syndrome is limited to a specific site, Xq28, and there is no generalized tendency to frequent DNA breaks or recombination. Although expression was modified by folic acid treatment in the patients, no consistent abnormality of folate metabolism could be identified.

Platelets and phospholipids in tissue factor-initiated thrombin generation.

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.

Effects of nutritional folate deficiency on the adhesive properties of murine melanoma cells.

Folate deficient murine B16 melanoma cells adhered more rapidly and in higher percentages to plastic plates or dishes coated with laminin or fibronectin than folate replete cells. These changes in the adhesive properties of murine melanoma cells induced by nutritional folate deficiency were not mediated by changes in cell size, proliferative capacity or cell cycle distribution. While melanoma cells served as a suitable surface for prothrombinase complex formation, folate deficiency did not alter this membrane function, suggesting that the membrane changes associated with folate deficiency are relatively specific.

Effects of pH on 5-methyltetrahydrofolic acid transport in human erythrocytes.

We measured the effects of external pH on influx, efflux and net steady-state levels for 14CH3H4PteGlu1. Initial rates of uptake were inversely proportional to external pH. Lowering the pH of the suspending medium increased influx by enhancing the affinity of the carrier: the apparent Km values at pH 6, 7.2 and 8.1 were 0.14, 0.25 and 0.63 microM respectively. In contrast, Vmax was independent of external pH. Efflux rate constants at pH 6, 7.5 and 8.5 were 0.034, 0.059 and 0.067/min respectively. consequently, lowering the external pH increased steady-state levels of 14CH3H4PteGlu1.

Immunosuppressive properties of chloroquinoxaline sulfonamide.

Chloroquinoxaline sulfonamide (CQS), a sulfanilamide derivative with antitumor activity, was found to be toxic to lymphoid tissue during preclinical studies. The mechanism of this toxicity appears to involve profound inhibition of lymphocyte activation. Incubation of human peripheral blood mononuclear cells (PBMNCs) with CQS decreased cellular incorporation of thymidine and deoxyuridine in a dose-dependent manner. Analysis of cell cycle distribution by flow cytometry indicated that CQS blocked movement out of the G0/G1 phase. Drug-treated cells were smaller and expressed fewer receptors for interleukin-2 (IL-2) and transferrin than untreated mitogen-stimulated lymphocytes. These observations support the notion that CQS has cell cycle specificity in regulating lymphocyte proliferation. As little as 10 microM CQS markedly inhibited both human lymphocyte and murine CTLL cell replication in response to IL-2 containing growth factors. However, CQS did not block secretion of IL-2 into culture supernatant fractions by human PBMNCs. Finally, CQS inhibited in vitro production of immunoglobulins G and M by mitogen-stimulated lymphocytes, primarily by causing cytotoxicity. In all of these drug effects, CQS was approximately one to two logs more potent than the parent compound, sulfaquinoxaline (SQ). These studies indicate that CQS inhibits essential basic processes in human lymphocytes. This agent may find use as an immunosuppressive drug.

Cellular pharmacology of chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma cells.

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.

Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1.

Mice developed massive splenomegaly and polyclonal hypergammaglobulinemia within 2 days after intravenous injection of a phosphorothioate oligomer that is antisense to a portion of the rev region of the HIV-1 genome. Histologic examination of spleens from injected animals showed marked expansion of a uniform-appearing population of small lymphocytes and many mitoses. Spleen mononuclear cells (SMNCs) from injected animals showed approximately a 10-fold-increased uptake of [3H]thymidine and production of IgM and IgG. Flow cytometry analysis indicated that the responding cells were predominantly B-lymphocytes. The anti-rev oligomer also was mitogenic in vitro and stimulated immunoglobulin production by normal mouse SMNCs and human peripheral blood mononuclear cells. Similar immunologic effects were observed with an anti-rev 21-mer phosphorothioate, truncated at the 3' end, but not with a 20-mer human p53 antisense phosphorothioate or a 28-mer anti-rev phosphodiester. These observations are consistent with the possibility that DNA sequences homologous to the rev gene participate in the regulation of mammalian lymphocyte activation, proliferation and maturation.

Inhibition of lymphocyte nucleic acid metabolism and antibody production by trimetrexate.

Trimetrexate is a lipid soluble dihydrofolate reductase inhibitor which, unlike methotrexate, does not depend upon the membrane folate transport system for cell entry. We investigated the possibility that trimetrexate (but not methotrexate) might permeate intermitotic lymphocytes and, following stimulation, impair only the responding cells, rather than all dividing cells, as is the case with methotrexate. Peripheral blood mononuclear cells from normal individuals were incubated for 1 hr in three moderate to high concentrations (1, 10 and 100 microM) of methotrexate or trimetrexate, washed, and incubated with phytohemagglutinin. Intracellular folate activity, as assessed by the deoxyuridine suppression test, was abnormal at all three concentrations of trimetrexate but only at the highest concentration of methotrexate. Similarly, incorporation of [3H]deoxyuridine was depressed profoundly in trimetrexate-treated cells (2% of control) but unaffected by methotrexate. Analysis of cell cycle distribution by flow cytometry confirmed G0 + G1 arrest in trimetrexate but not methotrexate-treated cells. Neither drug altered morphologic transformation, Tac antigen expression, or incorporation of [3H]thymidine by the "salvage" pathway. Therefore, brief exposure to methotrexate has little effect on intermitotic lymphocytes, whereas trimetrexate very specifically inhibits the conversion of deoxyuridine to thymidine in these cells and leads to the arrest of DNA synthesis in the G0 + G1 phase. This metabolic abnormality markedly reduces in vitro antibody synthesis: a 1-hr treatment of lymphocytes with 10 or 100 microM trimetrexate prior to incubation with pokeweed mitogen on four occasions completely inhibited both IgG and IgM secretion. Similar treatment with methotrexate had no effect until the highest concentration (100 microM) was used. We conclude that brief exposure of peripheral blood mononuclear cells to the nonclassical dihydrofolate reductase inhibitor, trimetrexate, results in inhibition of nucleic acid synthesis and impairment of antibody production. This drug effect may permit more incisive modulation of immune responses.

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)