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R Ashmun

Publications and source records attributed to R Ashmun.

8 recordsLinked to original sources
PubMed1999

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

3T3 Cells↗
PubMed1996

DNA index of glial tumors in children. Correlation with tumor grade and prognosis.

BACKGROUND: Although DNA index (DI) has prognostic significance in a variety of pediatric malignancies, there are few data regarding its utility in central nervous system (CNS) tumors. We have previously shown that patients with hyperdiploid medulloblastoma have a significantly better survival than those whose tumors are diploid. Here, we examine the effect of DI and tumor grade on the progression free survival (PFS) of 57 patients with a variety of glial neoplasms. METHODS: DI was determined by flow cytometry on freshly obtained tumor tissue from the initial diagnostic specimens; a DI = 1.0 was defined as diploid (DIP), 1.0 < DI < 1.1 as near diploid (NDIP), and DI > 1.1 as hyperdiploid (HYP). Tumors were histologically graded according to the World Health Organization classification. RESULTS: There were 21 Grade I tumors, 20 Grade II, 8 Grade III, and 8 Grade IV. Among the 41 low grade tumors (Grade I-II), 39 were DIP or NDIP, and 2 were HYP. Among the 16 high grade tumors (Grade III-IV), 9 were DIP, 2 NDIP, and 5 HYP. The 4-year PFS of low grade tumors was 70% (standard deviation [SD] 12%) versus 8% (SD 7%) for high grade tumors. There was a significant correlation between low grade tumor histology and a DIP/NDIP DI (P = 0.015), and univariate analysis suggested improved PFS was associated with DIP/NDIP tumors (P = 0.05). However, DI did not remain a significant prognostic factor after being stratified by tumor grade (P = 0.87). CONCLUSIONS: Unlike medulloblastoma, DI is not an independent prognostic factor in pediatric glial tumors.

Adolescent↗
PubMed1993

Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein.

Overexpression of bcl-2 delays the onset of apoptosis in lymphohematopoietic cells. We measured levels of bcl-2 protein in normal and leukemic human B-cell progenitors with a specific monoclonal antibody and flow cytometry. Normal immature B cells had low levels of bcl-2 protein; the intensity of fluorescence, expressed as molecules of soluble fluorochrome per cell, within CD10+ cells was 3,460 +/- 1,050 (mean +/- SD; 5 samples). In 16 cases of B-lineage acute lymphoblastic leukemia (ALL), cells had levels of bcl-2 that were strikingly higher than those of their normal counterparts (33,560 +/- 14,570; P < .001 by t-test analysis). We next investigated whether the intensity of bcl-2 expression correlated with the resistance of immature B cells to in vitro culture. In 12 cases of B-lineage ALL, the cells recovered after 7 days of culture on allogeneic bone marrow stromal layers were 69% to 178% (median, 95.5%) of those originally seeded. Prolonged survival of leukemic cells in vitro was observed even in the absence of stromal layers in 6 of these 12 cases; the intensity of bcl-2 protein expression in these cases was 45,000 +/- 13,270, compared with 21,500 +/- 7,260 in the 6 cases in which greater than 99.5% of cells rapidly died by apoptosis under the same culture conditions (P = .003). Five immature B-cell lines, continuously growing in the absence of stroma, had the highest bcl-2 expression (79,400 +/- 20,330). By contrast, most normal CD19+, sIg-immature B cells died despite the presence of bone marrow stromal layers; 9.7% to 28.2% were recovered after 7 days of culture in three experiments. We conclude that abnormal bcl-2 gene expression influences the survival ability of B-cell progenitors. This may contribute to leukemogenesis and explain the aptitude of leukemic lymphoblasts to expand outside the bone marrow microenvironment.

Adolescent↗
PubMed1991

Relationship of tumor-cell ploidy to histologic subtype and treatment outcome in children and adolescents with unresectable rhabdomyosarcoma.

Clinical and histopathologic features are often inadequate for accurate prediction of relapse or survival of individual patients with rhabdomyosarcoma (RMS). We therefore studied the cellular DNA content (ploidy) of RMS cells in relation to histology and response to therapy in 37 patients with unresectable tumors. Using flow cytometric techniques, we found that about one third of patients had diploid tumor stem lines, regardless of the histologic subtype. In the group with abnormal ploidy, a hyperdiploid classification (1.10 to 1.80 times the DNA content of normal diploid cells) was exclusively associated with embryonal histology (P = .001). By contrast, near-tetraploidy (1.80 to 2.60 times the DNA content of normal cells) was strongly associated with alveolar histology (P = .001). Thus, in these histologic subtypes of RMS, abnormal ploidy appears to arise through different mechanisms. Tumor-cell ploidy had a significant impact on survival that was especially apparent in patients with unresectable, nonmetastatic (group III) tumors. In this subgroup, hyperdiploidy conferred the best prognosis and diploidy the worst (P less than .0001). None of the eight patients with diploid tumors survived for more than 18 months. Tumor-cell ploidy was the best predictor of treatment outcome for patients with either embryonal (P less than .001; relative risk, 25.5) or alveolar (P = .073; relative risk 7.1) RMS and contributed significantly after adjustment for disease stage and anatomic site. Patients with unresectable diploid RMS have an unacceptably high risk of treatment failure, justifying new therapeutic approaches for this distinct subgroup.

Adolescent↗
PubMed1990

Relationship of deoxynucleotide changes to inhibition of DNA synthesis induced by the antiretroviral agent 3'-azido-3'-deoxythymidine and release of its monophosphate by human lymphoid cells (CCRF-CEM).

The metabolism and cytostatic effects of 3'-azido-3'-deoxythymidine (AZT), one of the most effective agents being used in the treatment of acquired immunodeficiency syndrome, were investigated in the CCRF-CEM line of human T lymphoid cells. The concentration of drug required to inhibit cell growth by 50% (CD50) was significantly lower when the cells were exposed to AZT for 24 hr (CD50 = 50 microM), as compared with 48 or 96 hr (CD50 = 225 and greater than 300 microM, respectively). AZT at 25 microM blocked the progression of cells in S phase for about 12 hr, but this effect was reversed by 24 hr, despite the continued presence of drug in the medium. At this drug concentration, the level of dTTP decreased to about 75% of the control level by 4 hr but rebounded to 30% above normal by 8 hr of drug exposure. dGTP and dATP pool sizes were unchanged, whereas the dCTP pool increased 5-fold. The time course of these biochemical changes indicated that the onset of S phase arrest was not directly related to the decrease in deoxynucleoside triphosphate pools. CCRF-CEM cells incubated with 25 microM AZT accumulated about 0.9 mM 5'-monophosphate (AZTMP) after 4 hr whereas levels of the 5'-di- and 5'-triphosphates (AZTDP and AZTTP) plateaued at about 2 and 5 microM, respectively. After this period, there was a rapid decrease in AZTMP accumulation, to one third its initial level by 24 hr, whereas AZTDP and AZTTP pools decreased to only about 70%. The loss in AZT nucleotide formation with time of drug exposure was associated with a concomitant accumulation of AZTMP in the medium. Cellular excretion of AZTMP was not associated with any detectable cell lysis or leakage of other cellular metabolites. The ability of CCRF-CEM cells to excrete AZTMP may be an important factor limiting the biochemical and biological effects of the drug.

Cell Division↗
PubMed1987

Expressions of resistance and cross-resistance in teniposide-resistant L1210 cells.

Resistance to teniposide (VM-26) by VM-26 selected resistant L1210 cells in culture was attributed to alterations in the flux of VM-26 across the plasma membrane and to functions of homogeneously staining regions that appeared on one or more chromosomes. In the present study, electrophoresis of membrane-cytosol fractions of these resistant sublines demonstrated a protein band, Mr 22 kd, that was not evident in similar fractions of drug-sensitive L1210 cells or three revertant sublines. The distribution of this protein among various cellular fractions could be altered by manipulation of the concentration of calcium ions. A representative subline, LIa5 microM, was observed to have vesicles that reacted with Sudan black B stain, an indication of altered lipid metabolism. The LIa5 microM subline was cross-resistant to etoposide, vincristine, doxorubicin, amsacrine, and actinomycin D. Concentrations of VM-26 that inhibited cell division to the same extent caused an accumulation of fewer cells in the G2 stage of cell division in LIa5 microM cultures than in L1210 cultures. These observations indicate that the LIa5 microM subline expressed multiple drug resistance, as well as changes in the expression of cytotoxicity to VM-26.

Animals↗
PubMedDate not supplied

Rhodamine 123 and flow cytometry to monitor the cytotoxic actions of nucleoside analogues in nondividing human lymphocytes.

We used human lymphocytes from the peripheral blood of normal individuals to compare the cytolytic actions of 2-chloro-2'-deoxyadenosine (2-Cl-dAdo) and 9-beta-D-arabinofuranosylguanine (ara-G). Membrane integrity was ascertained by flow cytometric quantification of the cells' uptake of the fluorescent mitochondria-specific probe rhodamine 123. Addition of 10 microM ara-G to lymphocyte cultures led to a progressive decline in the number of cells that could assimilate rhodamine 123, and after 2 days exposure to drug less than 50% of the cells showed normal staining compared to untreated cells. 2-Cl-dAdo had similar cytotoxic effects at a 0.1 microM concentration. Under the same conditions trypan blue revealed only a 10-20% toxic effect by these drugs. Using rhodamine 123 in combination with the vital stain propidium iodide, we found that 90% of the cells with abnormal rhodamine 123 uptake also stained with propidium iodide. The changes in membrane permeability to fluorescent dyes correlated with the loss of lymphocyte ability to respond to the plant mitogen phytohemagglutinin. Our data indicate that rhodamine 123 is a more sensitive probe than trypan blue for ascertaining early loss of the membrane integrity of dying lymphocytes. Combined with flow cytometry, rhodamine 123 uptake represents a simple and rapid method for identifying the important biochemical events associated with the cytocidal and cytostatic effect of drugs against normal and leukemic cells.

Arabinonucleosides↗