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Biomedical subjects

J L Cleveland

Publications and source records attributed to J L Cleveland.

At least 19 recordsLinked to original sources
PubMed2001

Mpl ligand prevents lethal myelosuppression by inhibiting p53-dependent apoptosis.

A single dose of Mpl ligand (Mpl-L) given immediately after lethal DNA-damaging regimens prevents the death of mice. However, the mechanism of this myeloprotection is unknown. The induction of p53-dependent apoptosis in response to DNA damage signals suggests that immediate administration of Mpl-L may inhibit p53-dependent apoptosis. This hypothesis was tested by administering a single injection of pegylated murine Megakaryocyte Growth and Development Factor (PEG-rmMGDF, a truncated recombinant Mpl-L) to p53(-/-) and wild-type mice immediately after carboplatin (80 mg/kg) and 7.5 Gy total body gamma-irradiation. PEG-rmMGDF was required to prevent the death of wild-type mice, whereas p53(-/-) mice survived with or without the exogenous cytokine. The degree of platelet depression and subsequent recovery was comparable in p53(-/-) mice to wild-type animals given PEG-rmMGDF. Hence, either Mpl-L administration or p53-deficiency protected multipotent hematopoietic progenitors and committed megakaryocyte precursors. The myelosuppressive regimen induced expression of p53 and the p53 target, p21(Cipl) in wild-type bone marrow, indicating that Mpl-L acts downstream of p53 to prevent apoptosis. Constitutive expression of the proapoptotic protein Bax, was not further increased. Bax(-/-) mice survived the lethal regimen only when given PEG-rmMGDF; however, these Bax(-/-) mice showed more rapid hematopoietic recovery than did identically-treated wild-type mice. Therefore, administration of Mpl-L immediately after myelosuppressive chemotherapy or preparatory regimens for autologous bone marrow transplantation should prevent p53-dependent apoptosis, decrease myelosuppression, and reduce the need for platelet transfusions.

Animals↗
PubMed2001

Suppression of c-myc expression and c-Myc function in response to sustained DNA damage in MCF-7 breast tumor cells.

The topoisomerase II inhibitors teniposide (VM-26), doxorubicin, and amsacrine (m-AMSA), as well as ionizing radiation, induce a transient suppression of c-myc mRNA, which correlates with growth inhibition of MCF-7 breast tumor cells. To further assess the involvement of c-mvc in the DNA damage-induced signal transduction pathways of the breast tumor cell, we determined the influence of sustained DNA damage on c-myc expression, c-Myc protein levels and c-Myc function. Continuous exposure of MCF-7 breast tumor cells to VM-26 induced DNA strand breaks that were sustained for at least 9 hr. DNA strand breakage was accompanied by a decline in c-myc transcripts and c-Myc protein levels by >90% after VM-26 exposure for 24 hr. The activity of a transcriptional target of the c-Myc protein, ornithine decarboxylase, was reduced by approximately 75% within 9 hr of DNA damage, in parallel to the declines in c-myc mRNA and protein levels. Extended exposure to VM-26 resulted in an initial loss of approximately 35% of the cell population followed by the death of additional cells such that by 72 hr only 50% of the cells were viable. Although apoptosis was evident 72 hr after initiating drug exposure [based on cell cycle analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and an assessment of cell morphology], the primary phase of cell killing, which occurred during the first 24 hr was non-apoptotic. These studies indicate that non-apoptotic pathways can also mediate cell death in the breast tumor cell and support the role of c-myc expression, c-Myc protein, and c-Myc function as elements of the DNA damage response pathway in the breast tumor cell.

Antineoplastic Agents↗
PubMed2001

c-Myc-mediated regulation of telomerase activity is disabled in immortalized cells.

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), as ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53.

3T3 Cells↗
PubMed2001

E2F-1 induces the stabilization of p53 but blocks p53-mediated transactivation.

E2F-1 induces p53 accumulation and E2F-1 and p53 form a physical complex, which affects the ability of E2F-1 to activate transcription. We mapped the domains on E2F-1 that interact with p53 and found two p53-binding domains. To understand the functional consequences of the E2F-1/p53 association on p53 activities we identified the domains of E2F-1 that were responsible for the accumulation of p53. Unexpectedly, we found that the E2F-1 transactivation domain was dispensable for p53 induction. By contrast, further deletion of the DP-1 interaction/'marked' box domain eliminated p53 accumulation. Radiolabeling pulse/chase analysis demonstrated that E2F-1 caused post-translational stabilization of p53. Although E2F-1 caused the stabilization of p53, E2F-1 expression impaired p53-dependent transactivation. Thus, the E2F-1 : p53 interaction may provide a checkpoint function to inactivate overactive E2F-1, but the association may also inactivate p53 transactivation to allow cell cycle progression.

Animals↗
PubMed2001

Circulating concentrations of IGF-I and IGFBP-3 are not predictive of incidence or clinical behavior of pediatric osteosarcoma.

BACKGROUND: Preclinical studies suggest a role of insulin-like growth factor-1 (IGF-1) in the proliferation of osteosarcoma cells in vivo. The purpose of this study is to address the relationship between serum levels of IGF-1 and its binding protein (IGFBP-3), and the clinical behavior and outcome of osteosarcoma in children, and to compare those levels present in osteosarcoma patients with a normal population. PROCEDURE: Serum IGF-1 and IGFBP-3 levels were determined by ELISA in 37 patients with osteosarcoma treated on the same treatment regimen (OS-91 protocol), and who had available serum samples from diagnosis. IGF-1 and IGFBP-3 levels were compared with those previously established in the normal population, matched for age and gender, and were correlated with the presence of metastatic disease, histologic response to preoperative chemotherapy, and event-free survival. RESULTS: In osteosarcoma patients the median IGF-1 level was 275 ng/ml (range, 105-613) and the median IGFBP-3 level was 3.4 mg/L (range, 2.3-5.1). IGF-1 levels differed from those in the normal population (P = 0.029); although we anticipated higher IGF-1 levels than normal children, 68% of observed standardized scores were less than 0. Furthermore, IGF-1 or IGFBP-3 levels failed to correlate with the presence of metastatic disease (P = 0.12 and P = 0.12, respectively), histologic response (Rosen-Huvos grades 3/4 vs. grades 1/2) (P = 0.95 and P = 0.71, respectively), or event-free survival (P = 0.52 and P = 0.41, respectively). There was a strong association observed between IGF-1 and IGFBP-3 levels (P < 0.001). CONCLUSIONS: In this retrospective study of 37 patients, we found that circulating levels of IGF-1 and IGFBP-3 are not predictive of the development or clinical characteristics of pediatric osteosarcoma. However, further studies on a larger patient population should be performed in order to investigate this relationship.

Adolescent↗
PubMed2001

Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis.

Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

ADP-Ribosylation Factor 1↗
PubMed2001

The ornithine decarboxylase gene is essential for cell survival during early murine development.

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Animals↗
PubMed2001

Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development.

Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

Animals↗
PubMed2000

Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation.

GSK-3beta-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3beta could inhibit nuclear import. Here, we report that GSK-3beta-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3beta, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division.

Active Transport, Cell Nucleus↗
PubMed2000

Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis.

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explanted DMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19(ARF), Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARF and functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null and ARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1(+/+) animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

3T3 Cells↗
PubMed2000

Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis.

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.

Animals↗
PubMed1999

Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis.

Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Emu) develop B-cell lymphoma and exhibit a mean survival time of approximately 6 months. The protracted latent period before the onset of frank disease likely reflects the ability of c-Myc to induce a p53-dependent apoptotic program that initially protects animals against tumor formation but is disabled when overtly malignant cells emerge. In cultured primary mouse embryo fibroblasts, c-Myc activates the p19(ARF)-Mdm2-p53 tumor suppressor pathway, enhancing p53-dependent apoptosis but ultimately selecting for surviving immortalized cells that have sustained either p53 mutation or biallelic ARF deletion. Here we report that p53 and ARF also potentiate Myc-induced apoptosis in primary pre-B-cell cultures, and that spontaneous inactivation of the ARF-Mdm2-p53 pathway occurs frequently in tumors arising in Emu-myc transgenic mice. Many Emu-myc lymphomas sustained either p53 (28%) or ARF (24%) loss of function, whereas Mdm2 levels were elevated in others. Its overexpression in some tumors lacking p53 function raises the possibility that Mdm2 can contribute to lymphomagenesis by interacting with other targets. Emu-myc transgenic mice hemizygous for ARF displayed accelerated disease (11-week mean survival), and 80% of these tumors lost the wild-type ARF allele. All ARF-null Emu-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Emu-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, cancelling its protective checkpoint function and accelerating progression to malignancy.

Animals↗
PubMed1999

The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects.

Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.

Animals↗
PubMed1999

Neuroprotection by a caspase inhibitor in acute bacterial meningitis.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Amino Acid Chloromethyl Ketones↗