Kiran Kaur

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long–term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell–cycle checkpoints and karyotypic stability in telomerase–expressing cells are similar to those of untransfected controls. In addition, co–expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.

Ral GTPases contribute to regulation of cyclin D1 through activation of NF-κB

ABSTRACT Ral GTPases have been implicated as mediators of Ras-induced signal transduction from observations that Ral-specific guanine nucleotide exchange factors associate with Ras and are activated by Ras. The cellular role of Ral family proteins is unclear, as is the contribution that Ral may make to Ras-dependent signaling. Here we show that expression of activated Ral in quiescent rodent fibroblasts is sufficient to induce activation of NF-κB-dependent gene expression and cyclin D1 transcription, two key convergence points for mitogenic and survival signaling. The regulation of cyclin D1 transcription by Ral is dependent on NF-κB activation and is mediated through an NF-κB binding site in the cyclin D1 promoter. Ral activation of these responses is likely through an as yet uncharacterized effector pathway, as we find activation of NF-κB and the cyclin D1 promoter by Ral is independent of association of Ral with active phospholipase D1 or Ral-binding protein 1, two proteins proposed to mediate Ral function in cells.

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.

Isolation of effector-selective ras mutants by yeast two-hybrid screening

Ras mutants displaying selective target interactions will often display partial loss of function phenotypes when expressed in cells. Thus the isolation of mutations in Ras and Ras family members has proved to be a productive approach for testing the requirement of specific target interactions to mediate downstream responses. The procedures outlined here greatly simplify the isolation of such mutants, and it is hoped will contribute to a better understanding of Ras effector function.

Adenine Phosphoribosyltransferase-Deficient Leishmania Donovani

Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (APRTase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of APRTase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wildtype levels of APRTase activity. These cells were reselected and colonies totally deficient in APRTase were isolated. Partially and totally APRTase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wildtype and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-HGPRTase pathway plays an important role in promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified APRTase and isoelectric focusing of crude extracts from wildtype and partially APRTase-deficient cells suggested that the latter cells possessed wildtype APRTase activity at half the amount found in wildtype parental cells. These data suggest that Leishmania donovani possess two copies of the APRTase structural gene and that these organisms might be diploid for the APRTase locus.

ISOLATION AND CHARACTERIZATION OF LEISHMANIA DONOVANI RESISTANT TO 5-FLUOROPYRIMIDINES: 100

From a mutagenized population of wildtype Leishmania donovani promastigotes, clonal isolates of mutant organisms were isolated in semi-solid agar containing 200μM 5-fluorouracil. These mutant organisms were cross-resistant to the cytotoxic effects of 5-fluorouridine but were just as sensitive to 5-fluorodeoxyuridine and methotrexate. Wildtype and mutant organisms, however, were equally capable of accumulating radiolabelled 5-fluorouracil, uracil, and uridine into anionic metabolites, suggesting a lack of a metabolic defect in the mutant organisms. Measurements of the intracellular nucleoside triphosphate levels did not indicate any abnormalities. Yet overnite incubations of wildtype cells with cytotoxic (50μM) concentrations of 5-fluorouracil markedly depleted pyrimidine nucleoside triphosphate pools without affecting ATP and GTP levels. In mutant organisms no such depletion of CTP and UTP were observed after similar incubations with 5-fluorouracil. Certain naturally occurring pyrimidines could protect wildtype cells from the cytotoxic effects of 5-fluorouracil. This biochemical genetic analysis of 5-fluorouracil-resistant Leishmania donovani suggests that 5-fluorouracil exerts its cytotoxic effects by inhibiting pyrimidine biosynthesis and that the mutant cells possess a genetically altered pyrimidine biosynthetic pathway.