Deficient Clones Research Articles

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

Biologic roles of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins.

The biologic functions of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins (type I and IV collagens, fibronectin, and laminin) were investigated by using the G(D3)-deficient mutant clone (SK-MEL-28-N1) and the parent cell line SK-MEL28. SK-MEL-28-N1 (N1) (high G(M3) expression: G(M3), 97.3%; G(D3), 0%) was selected by treating SK-MEL-28 (high G(D3) but low G(M3): G(M3), 6.5%, G(D3), 93.5%) with an anti-G(D3) monoclonal antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells. The N1 clone showed significantly higher ability to adhere to type I and IV collagens and laminin than the parent clone SK-MEL-28. In the N1 clone, the expression of alpha2beta1 and alpha3beta1 integrin receptors was increased, whereas in SK-MEL-28, their expression was very low or undetectable. The treatment with monoclonal antibodies directed specifically to G(D3) expressed on SK-MEL-28 inhibited the cell attachment to type IV collagen (33% inhibition of control), fibronectin (59%), and laminin (71%). These findings suggest that gangliosides G(M3) (by influencing integrin receptor levels) and G(D3) (by interacting directly with matrix proteins) might play some functional roles in attachment to extracellular matrix proteins and thereby enhance the metastatic potency of melanoma cells.

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Lymphocyte Subset Analysis and Glycosylphosphatidylinositol Phenotype in Patients With Paroxysmal Nocturnal Hemoglobinuria

Using multicolor flow-cytometry we have examined 19 patients with paroxysmal nocturnal hemoglobinuria (PNH) (18 with active disease and 1 spontaneous remitter) to determine absolute numbers of lymphocyte subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones amongst these subpopulations. Lymphocyte subsets were abnormal in all patients; the most frequent findings were low absolute numbers of natural killer (NK) cells (median, 0.08 x 10(9)/L; normal range, 0.2 to 0.4 x 10(9)/L) and low absolute numbers of B cells (median, 0.05 x 10(9)/L; normal range, 0.06 to 0.65 x 10(9)/L). GPI-deficient B, T, and NK cells were identified in 88%, 84%, and 89% of patients, respectively. The proportion of GPI-deficient cells within individual lymphoid lineages was highly variable, though in most patients the percentage of GPI-deficient NK cells was considerably higher than B or T cells. These observations can be explained when mechanisms of normal lymphopoiesis are considered. Despite these quantitative and qualitative abnormalities, no patients suffered an excessive number or severity of infections. The detection of PNH clones amongst all lymphocyte lineages may provide important information regarding the natural history of the disease and additional insights into kinetics of adult lymphopoiesis.

Lymphocyte Subset Analysis and Glycosylphosphatidylinositol Phenotype in Patients With Paroxysmal Nocturnal Hemoglobinuria

Using multicolor flow-cytometry we have examined 19 patients with paroxysmal nocturnal hemoglobinuria (PNH) (18 with active disease and 1 spontaneous remitter) to determine absolute numbers of lymphocyte subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones amongst these subpopulations. Lymphocyte subsets were abnormal in all patients; the most frequent findings were low absolute numbers of natural killer (NK) cells (median, 0.08 × 109/L; normal range, 0.2 to 0.4 × 109/L) and low absolute numbers of B cells (median, 0.05 × 109/L; normal range, 0.06 to 0.65 × 109/L). GPI-deficient B, T, and NK cells were identified in 88%, 84%, and 89% of patients, respectively. The proportion of GPI-deficient cells within individual lymphoid lineages was highly variable, though in most patients the percentage of GPI-deficient NK cells was considerably higher than B or T cells. These observations can be explained when mechanisms of normal lymphopoiesis are considered. Despite these quantitative and qualitative abnormalities, no patients suffered an excessive number or severity of infections. The detection of PNH clones amongst all lymphocyte lineages may provide important information regarding the natural history of the disease and additional insights into kinetics of adult lymphopoiesis.© 1998 by The American Society of Hematology.

Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with Rev-independent clones of human immunodeficiency virus type 1.

The posttranscriptional control element CTE of the simian type D retrovirus has been shown to support replication of Rev-Rev-responsive-element (RRE)-deficient molecular clones of human immunodeficiency virus type 1 (HIV-1). Upon infection of peripheral blood mononuclear cells in vitro, these CTE-containing Rev-independent viruses that are nef+ or nef-minus showed lower replicative capacity and infectivity than the wild-type HIV-1. We studied the effects of Rev-RRE replacement by the CTE on HIV-1 expression with SCID-hu mice. The nef+ and nef-minus Rev-independent viruses established infection with kinetics slower than that of the nef-minus NL4-3. Most importantly, no depletion of CD4-bearing thymocytes was observed after 6 weeks for mice infected with these Rev-independent viruses. This is in contrast to the infection with both wild-type and nef-minus viruses, which led to varying depletion of thymocytes. These data suggest an attenuated phenotype for growth and cytotoxicity of the Rev-independent HIV-1 clones in SCID-hu mice, independent of the presence of Nef. The mutant viruses, which have the essential Rev-RRE regulatory system eliminated, display a distinct phenotype not previously observed with HIV mutant viruses having deletions of accessory genes. Therefore, replacement of the Rev-RRE regulatory axis may generate viruses with altered biological properties in vivo.

Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function.

Mitochondria have been implicated in apoptosis, however, the precise mechanisms whereby mitochondria exert their effect are not clear. To gain further insights, we generated a panel of cells from ML-1a cells that were rendered respiration deficient by ethidium bromide treatment. Two respiration-deficient clones were subsequently reconstituted by fusion with platelets. Respiration-deficient clones were resistant to TNF-induced apoptosis, whereas ML-1a and reconstituted clones were sensitive. In contrast, inhibition of proliferation and induction of differentiation by TNF were still observed in respiration deficient clones, suggesting a selective requirement of respiration in TNF-induced apoptosis. Furthermore the apoptosis machinery is not completely altered in respiration-deficient cells because they underwent apoptosis after staurosporine treatment. Next, we showed that apoptosis induced by TNF and staurosporine were blocked by z-DEVD-CH2F, an inhibitor of CPP32-like cysteine protease, suggesting the involvement of CPP32-like protease in both apoptosis signaling pathways. Interestingly, TNF activated CPP32-like protease in the parental and reconstituted clones but not in respiration-deficient clones, and staurosporine in all clones. Thus, the apoptosis signaling block in respiration-deficient clones is located at a step before CPP32-like protease activation, which can be bypassed by staurosporine.

Impaired Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria/Aplastic Anemia Is Not Associated With a Selective Proliferative Defect in the Glycosylphosphatidylinositol-Anchored Protein-Deficient Clone

AbstractParoxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI− phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+CD59+ and CD34+CD59− cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+CD59− cells were both CD59− and CD55−. A very small population of CD34+CD59− cells was also detected in some normal volunteers; after sorting, these CD34+CD59− cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A–deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein–deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A– clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.

Age dependent selection against HPRT deficient T lymphocytes in the HPRT ± heterozygous mouse

The fraction of HPRT deficient T lymphocytes was measured in the HPRT ± female mouse between birth and an age of about 2 years. The animals were the F1 offspring of the HPRT deficient strain 129MF1 and HPRT competent C57BL 6J -mice . T lymphocytes from spleen were cloned in vitro and HPRT deficient clones were detected by double-labeling with [ 3H]thymidine and [ 14C]hypoxanthine. During the first weeks of life the fraction of deficient lymphocytes sharply decreases from about 50% at birth to 10–30% at an age of 10 weeks. In adult animals the fraction of HPRT deficient T cells smoothly further decreases to values about 10% at 80–90 weeks. The equation λ(t) = [0.547 × exp(−0.405 × t)] + [0.453 × exp(−0.0116 × t)] was found to be a good approximation of the time course of HPRT deficient cells in spleen; λ(t) is the fraction of deficient cells per competent cell and t is the age of animals in weeks. It is postulated that the selection against HPRT deficient T lymphocytes is the consequence of the reduced proliferative capacity of HPRT deficient cells (= selection factor). The time course of the ratio of deficient cells can be described as a function of the proliferation rate of the HPRT competent T cells and this selection factor. The sharp initial decrease is explained by a high selection pressure against HPRT deficient cells in young animals when the proliferation rate of the expanding T cell population is high and when T cells proliferate in the bone marrow. In adult animals the selection pressure against HPRT deficient cells is reduced, since T cells arise in general in peripheral lymphatic organs, where the salvage pathway is of lesser importance compared to the de novo purine synthesis. Implications of the selection against HPRT deficient lymphocytes for the widely used HPRT mutation assay are discussed.

Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin.

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.

DNA fingerprint analysis in chemically mutagenized Chinese hamster lung cells

Using a multi-locus minisatellite Per-6 DNA probe, we performed DNA fingerprint analysis. Chinese hamster lung (CHL) cells were treated with six model chemicals: N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methyl methanesulfonate, furylfuramide, 2-acetylamino-fluorene, and cyclophosphamide, with or without S9 mix. 771 hypoxanthine phosphoribosyltransferase deficient clones (749 from mutagen-treated cells and 22 from untreated cells) and 90 unselected clones from untreated cells were isolated and analyzed. The spontaneous mutation frequency at CHL cell minisatellite loci was 0.31-0.63%. All the chemicals increased mutation frequencies. Almost all mutations localized to the three specific minisatellite loci corresponding to 4.2, 3.8, and 2.4 kb bands, suggesting that these regions are more unstable and susceptible to mutation. DNA fingerprint analysis is a promising technique for detecting mutations at neutral DNA regions, especially recombinational mutations, and may be useful for surveying genetic instability related to heritable defects or aging.

S13.10 Generation of normal and glycosylphosphatidyl inositol (GPI)-deficient T-lymphocyte clones from a patient with paroxysmal nocturnal hemoglobinuria

S13.10 Generation of normal and glycosylphosphatidyl inositol (GPI)-deficient T-lymphocyte clones from a patient with paroxysmal nocturnal hemoglobinuria

Molecular analysis of mutations induced by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen and UVA in the mouse HPRT gene

The effects of the reaction photosensitized by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) on a mouse lymphoma cell line have been examined. Using the hypoxanthine phosphoribosyltransferase (HPRT) locus as target gene, a mutagenic effect of the photoreaction can be detected concomitantly with a loss of cell viability. Isolation of HPRT deficient clones has permitted a molecular characterization of the mutational pattern induced by the photosensitization reaction mediated by HMT. Southern blotting analysis demonstrated that the HPRT deficiency could not be correlated with gene deletions larger than 300 bp. Using polymerase chain reaction on both DNA and cDNA, amplification products have been cloned into M13mp18 and sequenced. Base transversions targeted on thymine residues have been located in exon 2, 3, 8 and 9 together with spontaneous frameshift mutations occurring in a run of guanine residues in exon 3. HPRT deficiencies owing to mutations arising in the HPRT promoter region have also been observed. Dot and Northern blot analysis revealed that the photoreaction could lead to either a reduced level of gene transcription or to a complete absence of HPRT m-RNA. Using polymerase chain reaction (PCR) amplification and agarose gel electrophoresis, deletions in the HPRT promoter have been observed and correlated to deficient enzyme expression.

Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK −) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK − or TK-proficient (TK +) cells was examined. As a result, the growth of TK − cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK + cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.

The molecular nature of spontaneous and chemically induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene in rat liver epithelial cells

The molecular nature of mutations in 6-thioguanine-resistant hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient clones of an adult rat liver (ARL) epithelial cell line mutated by benzo[ a]pyrene or aflatoxin B 1 was studied. DNA from these clones or spontaneous HGPRT-deficient mutants was subjected to Southern blotting using an HGPRT probe following DNA digestion with the restriction enzymes BamH1, EcoR1, HindIII or XbaI. With either the chemically induced or spontaneous mutants, no difference in restriction fragment pattern was observed between any of the mutants and their wild-type parent. However, differences were found between two lines ARL 6 and ARL 14 and the lines ARL 18, ARL 19 and DNA from Fischer rat hepatocytes. Although the variants did not display loss of HGPRT activity. It is suggested that deletion or loss of a pseudogene sequence could be the basis for the alterations in restriction fragment patterns.

5′-DEOXY–5′-METHYLTHIOADENOSINE (MTA) PHOSPHORYLASE DEFICIENCY IN LEUKEMIA: GENETICS AND BIOCHEMICAL ASPECTS: 28

MTA is produced in eukaryotic cells during the synthesis of polyamines from decarboxylated S-adenosylmethlonine. The nucleoside is rapidly cleaved to adenine and methylthioribose-1-P by MTA phosphoryiase (MTAse). We have assigned the gene MTAP to chromosome 9pter->9q12 by enzymatic and electrophoretic analysis of somatic cell hybrids. All normal tissues and non-malginant cell lines contain MTAse. However, several human leukemic cell lines are deficient in the enzyme, and 5 patients with acute lymphoblastic leukemia (ALL) have been shown thus far to lack MTAse in their malignant cells. Karyotypic abnormalities involving fragile site 9p21 occur in ALL with lymphomatous clinical features. One of 5 such patients studied prospectively lacked MTAse in her leukemic cells but not in normal blood cells at remission. No inactive enzyme protein has been detected by immunoadsorption among 7 leukemic lines tested. The MTAse deficient cell lines excrete MTA up to 0.32 nmol/hr/mg protein. In mice, the growth of MTAse deficient mutant lymphoma cells (but not MTAse positive wild type cells) causes plasma MTA to rise from undetectable levels to > 800 nM pre-terminally. Assay of plasma or urine MTA may thus prove useful to screen leukemic patients for MTAse deficient malignant cell clones.

Use of a replica technique to isolate muscle cell lines defective in expressing the acetylcholine receptor.

We have isolated genetic variants of the C2 muscle cell line that are defective in expressing the acetylcholine receptor (AcChoR). Because the AcChoR is expressed only after C2 myoblasts have fused to form myotubes, we employed a replica technique to detect the variants. This technique yields two copies of each clone, one of which can be used for screening and the other, as a source of dividing cells. In a screening of about 10,000 clones derived from mutagenized cells, we found 2 that fused normally and expressed normal levels of acetylcholinesterase but had reduced amounts of AcChoR on their surface. One of these also had a reduced level of intracellular AcChoR, but, in the other, the amount of intracellular AcChoR was 5-fold higher than normal. Several variants were found that failed to fuse and had reduced levels of both AcChoR and acetylcholinesterase. Though we relied on 125I-labeled alpha-bungarotoxin to distinguish wild-type from deficient clones, we found that an antiserum to the AcChoR, followed by a biotinylated second antibody and a horseradish peroxidase-avidin complex, could also be used. Therefore, it should be possible to obtain muscle cell variants defective in the expression of a variety of proteins for which specific antibodies are available.

Identification of the nucleoside transporter in cultured mouse lymphoma cells. Photoaffinity labeling of plasma membrane-enriched fractions from nucleoside transport-competent (S49) and nucleoside transport-deficient (AE1) cells with [3H]nitrobenzylthioinosine.

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.

The study of human myeloid differentiation using bromodeoxyuridine (BrdU).

Little is known concerning the mechanism of myeloid differentiation. A human promyelocytic cell line (HL-60) differentiates to granulocytes or macrophage-like cells when cultured with a variety of agents. How these agents trigger myeloid differentiation is not understood. This study shows that 1.0-10.0 micrograms/ml bromodeoxyuridine (BrdU) induced myeloid differentiation of HL-60 in liquid culture. After 7 days, BrdU (3.0 micrograms/ml) produced only moderate inhibition of HL-60 growth, but induced myeloid maturation with 40% of the cells becoming morphologically more mature; 41% developed the ability to reduce nitroblue tetrazolium (NBT); 19% phagocytized Candida albicans; and 18% developed Fc receptors. The action of BrdU was mimicked by 5-iodo-deoxyuridine. Thymidine (Td) (1- to 10-fold excess) competitively inhibited incorporation of [3H]BrdU into DNA of HL-60 and inhibited the triggering of HL-60 differentiation by BrdU. The BrdU-induced maturation of HL-60 correlated with the incorporation of BrdU into DNA of HL-60. DNA buoyant density studies showed that about 46% of the Td was replaced by BrdU in each DNA strand of HL-60 as the cells differentiated in culture containing 3 micrograms/ml BrdU for 7 days. We established 20 thymidine kinase (TK)-deficient HL-60 clones. The HL-60 TK-deficient cells were unable to phosphorylate Td, to incorporate either [3H]Td or [3H]BrdU or differentiate in the presence of BrdU (1-1000 micrograms/ml). The HL-60 TK-deficient cells retained the ability to differentiate in the presence of other HL-60 inducers. Taken together, the studies suggest myeloid differentiation of HL-60 is triggered because of incorporation of BrdU into DNA of the cells.

UV sensitivity in thymidine kinase deficient mouse erythroleukaemia cells

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.