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Biomedical subjects

A Kimchi

Publications and source records attributed to A Kimchi.

At least 55 records · Page 3Linked to original sources
PubMed1993

Complementation by wild-type p53 of interleukin-6 effects on M1 cells: induction of cell cycle exit and cooperativity with c-myc suppression.

Stable transfection of M1 myeloid leukemia cells with a temperature-sensitive mutant of p53 results in two phenomena that are manifested exclusively at the permissive temperature. On one hand, activation of wild-type p53 by the temperature shift induced an apoptotic type of cell death which could be inhibited by interleukin-6 (IL-6) (E. Yonish-Rouach, D. Resnitzky, J. Lotem, L. Sachs, A. Kimchi, and M. Oren, Nature 352:345-347, 1991). On the other hand, as reported in this work, activated p53 complemented the antiproliferative effects of IL-6 in M1 cells. A shift to the permissive temperature concomitant with or early after IL-6 treatment imposed a novel pattern of cell cycle arrest in which about 95% of the cells were retained within a G0-like quiescent state. This phase was characterized by 2N DNA content and low RNA and protein content. On the molecular level, activation of wild-type p53 transrepressed the c-myc gene but not the cyclin A, D1, or D2 gene, which are all independently suppressed by IL-6 in M1 cells. To further analyze whether c-myc inhibition mediates or complements p53 effects, the p53-transfected M1 cells were infected with a retroviral vector expressing deregulated c-myc, refractory to p53 or IL-6 action. It was found that the process of cell death was not interrupted at all in these M1 c-myc-p53 double transfectants, suggesting that the transrepression of c-myc is not a major obligatory event mediating p53-induced cell death. In addition, some of the antiproliferative effects of activated p53, manifested in the presence of IL-6, could still be transmitted in the background of constitutive c-myc. Yet the context of deregulated c-myc interfered with the final accumulation of cells within a G0-like phase, suggesting complementary interactions between the outcome of p53 activation and of c-myc suppression in the control of cell cycle arrest.

Cell Cycle↗
PubMed1993

p53-mediated cell death: relationship to cell cycle control.

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.

Animals↗
PubMed1993

Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.

Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells.

Base Sequence↗
PubMed1993

Interleukin 6 induces DNA binding activity of AP1 in M1 myeloblastic cells but not in a growth resistant cell derivative.

The effects that three different growth inhibitory cytokines exert on expression and function of members of the Jun family were studied in this work. M1 myeloblastic cells were chosen for this purpose because of their high growth sensitivity to interleukin 6 (IL-6), transforming growth factor beta 1 and alpha- and beta-interferons. It is reported here that IL-6 elevated the junB and c-jun mRNA levels and induced the formation of a novel DNA-protein complex with high sequence specificity to 12-O-tetradecanoylphorbol-13-acetate response element (TRE) oligonucleotides. This IL-6 induced TRE binding complex was abolished by anti-Jun specific antibodies and was efficiently competed by an oligonucleotide that comprises the mouse homologue of a previously described human c-myc negative DNA element. It persisted in cells for at least 48 h after IL-6 treatment and failed to be induced by alpha- and beta-interferons or by transforming growth factor beta 1, which affected differently the pattern of jun mRNA expression. To further explore regulatory and functional aspects of this induced TRE binding activity, an IL-6 resistant M1 clone was isolated and further analyzed. This clone carried a postreceptor deficiency that abrogated completely the growth inhibitory responses to IL-6 but did not interfere with the induction of two differentiation related cell surface markers. Interestingly, the IL-6 resistant clone had lost two molecular responses to IL-6, induction of TRE binding activity and suppression of the c-myc gene. The data correlate the IL-6 induced AP1 activity with the suppression of c-myc and growth inhibition.

Animals↗
PubMed1992

Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G0/G1-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical G0/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G0/G1-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor beta, reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced G0/G1-specific arrest requires at least these two receptor-generated events.

Animals↗
PubMed1992

Cytokine triggered molecular pathways that control cell cycle arrest.

Recent progress has been made concerning the understanding of the molecular pathways that mediate the growth suppressive effects of inhibitory cytokines. Interferons, interleukin-6 and transforming growth factor-beta were investigated in these studies. Cell lines that display growth sensitivity to all three cytokines and growth resistant derivates provided a suitable genetic background to determine whether common or unique post-receptor elements mediate the effects of each cytokine. Three nuclear genes, c-myc, RB, and cyclin A were found to be common key downstream targets along the cytokine induced growth suppressive pathways. Genetic and pharmacological manipulations proved that these molecular responses fall into few complementary pathways that function in parallel to achieve the cytokine mediated G0/G1 arrest. New strategies, such as knock out anti-sense gene cloning were developed and they currently provide powerful tools for the isolation of genes along the signaling pathways of growth arrest.

Animals↗
PubMed1991

Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6.

Wild-type p53 protein has many properties consistent with its being the product of a tumour suppressor gene. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation as well as in mediating growth arrest by growth-inhibitory cytokines. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.

Animals↗
PubMed1991

A genetic tool used to identify thioredoxin as a mediator of a growth inhibitory signal.

Loss of sensitivity to growth inhibitory polypeptides is likely to be one of the events that participates in the formation of some tumors and might be caused by inactivation or loss of the genetic elements that transduce these extracellular signals. The isolation of such a gene was achieved by randomly inactivating genes by an anti-sense complementary DNA expression library followed by direct selection for growth in the presence of an inhibitory polypeptide. Thus, a gene whose inactivation conveyed growth resistance to interferon-gamma (IFN-gamma) was isolated. Sequence analysis showed complete identity with human thioredoxin, a dithiol reducing agent, implicated here in the IFN-gamma-mediated growth arrest of HeLa cells.

Blotting, Northern↗
PubMed1991

The antiproliferative effect of cyclosporine on hematopoietic and lymphoblastoid cell lines--common mechanistic elements with interferon-alpha.

Cyclosporine, but not its nonimmunosuppressive analog cyclosporine H (CsH), caused in a variety of hematopoietic cell types a growth arrest in the G0/G1 phase of the cell cycle. This arrest was associated with a significant reduction in the c-myc mRNA levels, which could be observed already 1 hr following CsA treatment. Similarity between the antiproliferative effects of CsA and IFN-alpha was observed. Thus, the IFN-alpha sensitive human B-lymphoblastoid cell line Daudi was also sensitive to CsA while an IFN-alpha resistant variant of Daudi cells was found to be resistant to CsA as well. Inhibition of protein synthesis with cycloheximide during IFN-alpha or CsA treatment blocked their ability to reduce the expression of c-myc. Depletion of protein kinase C (PKC) activity from cells by pretreatment of Daudi cells with phorbol.12-myristate 13-acetate (PMA) abolished the G0/G1 arrest induced by both CsA and IFN-alpha. Combinations of low concentrations of CsA and IFN-alpha had synergistic effects on cell-cycle distribution and on c-myc mRNA level, suggesting that CsA and IFN-alpha differ in some features of their antiproliferative action. This conclusion was supported by the observation that a CsA-resistant variant of Daudi cells was found to retain its sensitivity to IFN-alpha. In addition, reduction of ornithine decarboxylase mRNA expression was obtained with IFN-alpha but not with CsA. Taken together, our results suggest that CsA and IFN-alpha share some common element(s) in the pathways of their antiproliferative activity. The possible mechanisms of their antigrowth effects and the clinical significance of our findings are discussed.

Blotting, Northern↗
PubMed1991

Deregulated c-myc expression abrogates the interferon- and interleukin 6-mediated G0/G1 cell cycle arrest but not other inhibitory responses in M1 myeloblastic cells.

The proliferation of M1 myeloblastic cells can be specifically restricted at the G0/G1 phase of the cell cycle by exposure to alpha- and beta-interferons or to interleukin 6. The latter cytokine also induces the morphological and functional differentiation of these myeloblasts toward monocytes. Each of these two different cytokines suppresses the expression of the c-myc nuclear oncogene, and the selective reduction in c-myc mRNA and protein precedes the cell cycle changes. In order to investigate whether one or more of the growth-suppressive effects of interferon and interleukin 6 are mediated by c-myc reduction, M1 cells were transfected with SV40-driven c-myc plasmid, whose expression fails to be turned off by these two cytokines. A detailed analysis of the responses to interferon and to interleukin 6 revealed that all of the myc-transfected clones have lost the cytokine-mediated G0/G1 type of growth arrest. However, not all of the growth responses to these cytokines were rescued by this specific genetic manipulation, and the cytokine-treated transfected cells stopped to proliferate in a new fashion which was not cell cycle specific. In addition, the myc-transfected cells developed the differentiated phenotype in response to interleukin 6, as determined by the morphological change, expression of Fc receptors, and cytochemical analysis, suggesting that these molecular events can occur in the monocyte cell lineage in spite of the abnormal constitutive expression of c-myc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The involvement of protein kinase C in mediating growth suppressive signals of interferons in hematopoietic cells.

The possible involvement of protein kinase C in transducing the growth suppressive signals of interferons was studies in this work in two different hematopoietic cell lines. Chronic exposure of human Burkitt lymphoma and mouse M1 myeloblastic cell lines to phorbol myristate acetate (PMA), reduced by more than 90% the PKC protein levels and enzymatic activity in cell extracts. The depletion of PKC from cells abrogated the ability of IFN (alpha + beta) to arrest cell growth at the G0/G1 resting phase of the cell cycle. In contrast, other responses to IFN such as the induction of (2'-5') oligoadenylate synthetase gene, continued to take place at the same dose response pattern thus excluding the possibility that early targets in the pathway, such as the number or affinity of IFN cell surface receptors might be affected by PMA. The same prolonged treatment of M1 cells with PMA did not interfere with the ability of another cytokine, transforming growth factor beta (TGF-beta), to induce the normal type of G0/G1 arrest further supporting the specificity of the effect towards IFN responses. Unexpectedly, depletion of PKC from cells did not interfere with the negative effects of IFN on c-myc mRNA and protein expression in spite of the direct involvement of this molecular event in growth responses to IFN. The putative PKC dependent molecular event could therefore function either downstream to or in combination with the reduction in c-myc protein levels, providing a necessary but not a sufficient step to arrest cell cycle progression at the G0/G1 phase.

Animals↗
PubMed1988

Absence of TGF-beta receptors and growth inhibitory responses in retinoblastoma cells.

The responses of retinoblastoma tumor cells and normal retinal cells to various growth inhibitory factors were examined. Whereas fetal retinal cells were highly sensitive to the antimitogenic effects of transforming growth factor beta 1 (TGF-beta 1), retinoblastoma tumor cell lines were all resistant to this factor. Binding assays and affinity labeling of these cells with radioiodinated TGF-beta 1 revealed that the cells did not have TGF-beta receptors. The retinoblastoma cells lacked the three affinity-labeled proteins of 65, 95, and 300 kilodaltons typically seen in human cell lines and thus differed from normal retinal cells and from other types of neuroectodermal tumors that display the normal pattern of receptors. Loss of TGF-beta receptors, which is a rare event among tumor cells, may represent one mechanism through which these cells escape from negative control and form retinoblastomas.

Cell Division↗
PubMed1988

Recessive genetic deregulation abrogates c-myc suppression by interferon and is implicated in oncogenesis.

In a previous study we demonstrated that many hematopoietic tumor cells are resistant to the inhibitory effects that interferon exerts on c-myc mRNA expression without losing other receptor-mediated intracellular responses (M. Einat, D. Resnitzky, and A. Kimchi, Nature [London] 313:597-600). We report here that this partial resistance was overridden in two independent stable somatic cell hybrids prepared by fusion between sensitive and resistant cells. The c-myc mRNA transcribed from the active allele of the resistant parent cell was reduced by interferon within the context of the cell hybrid. It was therefore concluded that changes in the cis-acting sequences of c-myc were not involved in this type of relaxed regulation and that resistance resulted rather from inactivation or loss of postreceptor elements which operate in trans. The growth-stimulating effect that this genetic deregulation might have on cells was tested in experimental systems of cell differentiation in which an autocrine interferon is produced. For that purpose we isolated variant clones of M1 myeloid cells which were partially resistant to alpha and beta interferons and tested their growth behavior during in vitro-induced differentiation. The resistant clones displayed higher proliferative activity on days 2 and 3 of differentiation than did the sensitive clones, which stopped proliferating. The loss of c-myc responses to the self-produced interferon disrupted the normal cessation of growth during differentiation and therefore might lead cells along the pathway of neoplasia.

Burkitt Lymphoma↗
PubMed1988

Transfection of fibroblasts with activated c-myc confers resistance to antigrowth effects of interferon.

The Go/G1 to S transition in growth factor-stimulated Balb/c 3T3 fibroblasts is efficiently blocked by interferons (IFNs). In the present communication we studied whether deregulated expression of exogenously introduced c-myc changes the growth sensitivity to type I IFNs (alpha + beta). Constructs that link the two coding exons of c-myc to the long terminal repeat (LTR) of Ha-MS virus were introduced into the 3T3 fibroblasts. The steady state levels of exogenous c-myc mRNA in the individual stable clones were 3-10 fold higher than the endogenous mRNA levels in non transfected Balb/c 3T3 cells. The expression directed from the c-myc-construct was found to be completely resistant to inhibition by IFN while it was still partially responsive to addition or depletion of growth factors. To test the possible phenotypic changes after this genetic manipulation, the cell cycle distribution of individual c-myc-transfected clones was analyzed during the growth factor controlled transition from resting phase to proliferating state. We find that entry into S phase of the c-myc-transfected clones became completely resistant to inhibition by low IFN concentrations which blocked this process in the parental cell line and the control clones. It is concluded that deregulated expression of c-myc resulting from substitution of the authentic promoters and the first c-myc exon with a viral promoter reduces the sensitivity of synchronized fibroblasts to the antimitogenic effects of IFN.

Animals↗
PubMed1987

The clinical significance of exercise-induced left ventricular wall motion abnormality occurring at a low heart rate.

We studied the relationship between the heart rate at the time of onset of exercise-induced wall motion abnormality and the severity of coronary artery disease in 89 patients who underwent exercise equilibrium radionuclide ventriculography as part of their evaluation for coronary artery disease. Segmental wall motion was scored with a five-point system (3 = normal; -1 = dyskinesis); a decrease of one score defined the onset of wall motion abnormality. The onset of wall motion abnormality at less than or equal to 70% of maximal predicted heart rate had 100% predictive accuracy for coronary artery disease and higher sensitivity than the onset of ischemic ST segment depression at similar heart rate during exercise: 36% (25 of 69 patients with coronary disease) vs 19% (13 of 69 patients), p = 0.01. Wall motion abnormality occurring at less than or equal to 70% of maximal predicted heart rate was present in 49% of patients (23 of 47) with critical stenosis (greater than or equal to 90% luminal diameter narrowing), and in only 5% of patients (2 of 42) without such severe stenosis, p less than 0.001. The sensitivity of exercise-induced wall motion abnormality occurring at a low heart rate for the presence of severe coronary artery disease was similar to that of a deterioration in wall motion by more than two scores during exercise (49% vs 53%) or an absolute decrease of greater than or equal to 5% in exercise left ventricular ejection fraction (49% vs 45%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Changes in p53 mRNA expression during terminal differentiation of murine erythroleukemia cells.

The protein p53 is functionally implicated in the normal regulation of cell proliferation. We have previously reported that the rate of p53 protein synthesis is reduced during the cessation of cellular proliferation which accompanies the in vitro induced differentiation of Friend-erythroleukemia cells. In this work we followed the p53 mRNA expression during the differentiation of these cells. We report on a new type of p53 mRNA with a slower electrophoretic mobility on gels, which appeared in the cytoplasmic fraction of the erythroleukemia cells between 1 to 3 days following induction of differentiation and persisted in the cells until Day 7. The larger type of p53 mRNA was found associated with polysomes, suggesting that it is translatable in cells. The difference in size between the noninduced and the differentiation-specific type of p53 mRNAs (about 200 nucleotides) was not abrogated following the deadenylation of the mRNAs, thus excluding the possibility that the altered size might result from a longer poly(A) tract. S1 nuclease mapping of the 3' termini of the p53 mRNAs revealed that the 3' ends of both p53 mRNA types were identical, suggesting that either alternative splicing or a longer 5' noncoding region could cause this heterogeneity in p53 mRNA transcripts.

Animals↗