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J J Lawrence

Publications and source records attributed to J J Lawrence.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein.

We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with S100b, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and S100b was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with S100b-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperature-dependent oligomerization and that the interaction between S100b and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of S100b to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the G0-G1/S boundary.

Amino Acid Sequence↗
PubMed1992

Antisense RNA and p53 regulation in induced murine cell differentiation.

p53 expression is strongly modulated during the process of induced differentiation, at the same time as both cell cycle and genetic expression become modulated, giving rise to a commitment to terminal differentiation. We took advantage of two murine cell lines inducible for differentiation, an erythroleukemia and a melanoma cell line, to outline common features of the regulation of p53 expression during the differentiation process. We found that p53 mRNA decreased early after induced differentiation and that regulation was controlled at a posttranscriptional level. Our data showed that this regulation affects p53 pre-mRNA maturation. Because, in both systems used, actinomycin D treatment abolished the inducer-mediated decrease of p53 mRNA, we looked for induced RNAs potentially involved in this process. Using different parts of the p53 gene and flanking regions as probes, we identified three RNA species whose expression is modulated during induced differentiation. A first species is made of high molecular weight RNAs that accumulate in the nuclear compartment and seem to represent antisense transcripts of the p53 gene. A second species, 1.3-kb long, was found to accumulate in the nucleus of induced MEL cells and was homologous to a restricted part of the first intron of the p53 gene due to the presence of a B1 repetitive element in an antisense orientation with respect to the p53 pre-messenger RNA. Finally, a family of B2-containing RNAs was observed in both cytoplasmic and nuclear compartments. The variation in the amounts of sense and antisense RNAs, respectively, suggested an interesting speculative model for the maturation of B2-containing pre-messenger RNAs.

Animals↗
PubMed1992

Induction of H1(0)-gene expression in B16 murine melanoma cells.

Histone H1(0) accumulation is associated with the terminal stage of differentiation. Unlike other H1 histones, it is able to accumulate in the absence of DNA synthesis, however the transcription of its gene is cell-cycle dependent. The regulation of H1(0)-gene expression has been studied during the induced differentiation of B16 cells and during reversion of the process, which may be achieved when induced cells are released into an inducing-agent-free medium. During the earlier period of induced differentiation, H1(0) mRNA showed over-expression when the cells were still proliferating. Then the amount of H1(0) mRNA decreased as the cells became arrested in G0-G1. H1(0) mRNA half-life measurements and run-on experiments demonstrated that such modulation of the amount of mRNA originated from a transcriptional control of H1(0)-gene expression. When induced cells reverted to a proliferative undifferentiated state, H1(0) mRNA decreased very rapidly, indicating that an active process was involved in this decay. This behavior differed from that observed in rat liver hepatocytes allowed to proliferate and de-differentiate after partial hepatectomy, or in murine erythroleukemia cells when the inducing agent was removed from the culture.

Animals↗
PubMed1992

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation.

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results.

Acetamides↗
PubMed1991

Multiple control level governing H10 mRNA and protein accumulation.

We have studied the variation of histone H10 and of its coding mRNA during rat liver regeneration after partial hepatectomy. Our data showed that while H10 decreased when cell proliferation was initiated, H10 mRNA accumulated in a proliferation-dependent manner as did H3 mRNA. These results showed two interesting aspects of the regulation of H10 expression in vivo, confirming results we have obtained previously in vitro: first H10 mRNA accumulation is a proliferation-dependent event; second, H10 protein accumulation may be uncoupled from that of its coding mRNA.

Animals↗
PubMed1991

Modulation of B2 containing small RNAs during induced differentiation of murine erythroleukemia cells.

We identified a B2 repetitive element approximately 1.9kb down stream from mouse p53 coding gene. This element was then used as a probe to investigate the expression of B2 containing RNA during the induced differentiation of murine erythroleukemia (MEL) cells. This probe revealed two nuclear and one cytoplasmic RNA species. Nuclear small RNAs had a biphasic variation: a decrease followed by a reaccumulation. The cytoplasmic species was essentially non polysomal, and disappeared after the induced differentiation. The presented results suggest that the regulation of these RNAs is associated to cell proliferation and differentiation respectively.

Animals↗
PubMed1991

Regulation of histone H1(0) accumulation during induced differentiation of murine erythroleukemia cells.

Histone H1(0) is one of the potential candidates that may contribute to the onset and stabilization of a genetic program during induced differentiation of murine erythroleukemia cells. In an attempt to understand better the role of H1(0) in this process we have tried to determine at which level the regulation of its induced accumulation occurs. Protein H1(0) was found to increase by a factor of 3 while its mRNA increased by a factor of 14, due to activation of gene transcription. As shown by H1(0) half-life measurements, the difference between the actual amount of H1(0) and that expected from the amount of mRNA was not due to increased turnover of the protein. Fractionation of the translational apparatus at several times during induction, revealed that H1(0) mRNA was efficiently transferred to the high molecular weight polysomes. The rate of synthesis of H1(0) was also increased by a factor of 4. Taken together, these results suggest the existence of a strong control at the translational level, which regulates H1(0) accumulation.

Animals↗
PubMed1991

Cell cycle-related accumulation of H1(0) mRNA: induction in murine erythroleukemia cells.

H1(0), a member of histone H1 family, is associated with cell differentiation in vivo and in vitro. Unlike other histones its synthesis is known to be constitutive during the cell cycle. The present paper provides evidence showing that, in murine erythroleukemia (MEL) cells, H1(0) mRNA accumulation is coupled to DNA replication, but this mRNA is stable enough to remain present all over the cell cycle. Moreover the experiments suggest that H1(0) mRNA accumulates in mid or late S phase rather than when cells initiate DNA replication as does H3 mRNA. H1(0) gene transcription is drastically induced by chemical inducers of differentiation in this cell line. In an attempt to better understand the mechanism of this process, we have studied the inducibility of this gene with respect to the position of the cells in the cell cycle. The experiments show that the most efficient accumulation of H1(0) is observed when cells are treated by the inducer during the S phase of the cell cycle. In the same conditions, H3 mRNA accumulation remains unaffected. These results show that H1(0) mRNA, like H3 mRNA, accumulates during DNA replication, although at a different stage in this phase. The two genes differ, however, in their response to the inducer, suggesting that different regulatory elements might be involved in such a response.

Acetamides↗
PubMed1990

Recovery of RNA from flow-sorted fixed cells.

We describe a method for the preparation of RNA from ethanol-fixed cells, allowing analysis of the RNA from cells "frozen" in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations before RNA preparation, such as investigations of cell-cycle-regulated gene expression, which require the preparation of cells for cell-cycle flow analysis, and even for long-term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell-cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.

Animals↗
PubMed1989

An antisense RNA involved in p53 mRNA maturation in murine erythroleukemia cells induced to differentiate.

A post-transcriptional control of gene expression was found to be responsible for a down-regulation of p53 mRNA accompanying the induced differentiation of murine erythroleukemia (MEL) cells. Such a post-transcriptional control was governed by the induced synthesis of an RNA species (inRNA). In an attempt to find a potential candidate for such a function, we have localized the post-transcriptional regulation of p53 mRNA in the nuclear compartment of the cells; the various fragments of the p53 gene were used as probes for induced RNA(s) susceptible to interacting with p53 pre-mRNA. This experimental approach allowed for the identification of a nuclear RNA molecule, approximately 1.3 kb long, which was recognized specifically by a PstI-HindIII fragment located in the 5' part of the first intervening sequence of the p53 gene. This RNA accumulated when cell were treated by the inducer concomitantly with high mol.wt p53 mRNA precursors. However this RNA was not a maturation product of p53 pre-mRNA as evidenced by its antisense orientation with respect to this RNA. Moreover it was markedly enriched in the poly(A)+ fraction. The complementary part of inRNA in the p53 gene has been sequenced over approximately 1200 bp; no extensive homology was found in gene data banks but three restricted areas of the sequence were found homologous to a limited number of genes; they were themselves partially homologous to known repetitive sequences. Possible implication of such a sequence in the regulation of p53 gene expression is discussed.

Animals↗
PubMed1989

Flow cytofluorimetric determination of protein distribution throughout the cell cycle.

Quantitative treatment of multiparametric determination on cells using flow cytofluorometry was made possible by the development of a computer program that allows the relative quantification of a specific protein as a function of the position of cells during the cell cycle. This type of analysis provides interesting information about the distribution of a given protein throughout the cell cycle. Four examples showing the distribution of specific proteins illustrate such a quantification during the cell cycle in two different cell lines. The program also allows for the handling of a series of histograms obtained by the analysis of protein distribution as a function of DNA content in relation to a third parameter. To illustrate possible applications for this program, the evolution of the distribution of two proteins, the oncoprotein p53 and the histone H1(0), during the induced differentiation of murine erythroleukemia cells has been studied.

Animals↗
PubMed1989

Histone H1(0) mapping using monoclonal antibodies.

Monoclonal antibodies (mAb) to ox liver histone H1 degree were produced and characterized. Two sets of mice were immunized either with pure H1(0) or with an H1(0)-yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained kappa light chains. Immunoblotting experiments using peptides derived from H1(0) or H5 treated by various proteolytic agents (trypsin, N-bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1(0). More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24-27 and were specific for H1(0). Six mAb bound to residues 27-30 and were specific for H1(0) except one of them which strongly cross-reacted with H5 and GH5. Two mAb reacted with the entire histone H1(0) but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1(0) globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1(0) and the various IgG1 mAb by increasing amounts of peptide-bovine serum albumin conjugates were then performed.

Amino Acid Sequence↗
PubMed1988

Cell cycle regulation of poly(ADP-ribose) synthetase in FR3T3 cells.

The properties of poly(ADP-ribose) synthetase were studied throughout the cell cycle progression of non-synchronized rat FR3T3 fibroblasts using an immunological and biochemical approach. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population by using flow cytofluorometry. G1, S and G2 + M fractions were used for enzymatic assays in the presence of saturating concentrations of DNAase I for the analysis of total poly(ADP-ribose) synthetase; maximal enzyme activity was found in the G2 + M phase. Purified IgG, specific for the FR3T3 poly(ADP-ribose) synthetase were used for the labelling of endogenous synthetase in order to quantify the enzyme immunologically. Localization of nuclear immunofluorescence was observed and analysis of poly(ADP-ribose) synthetase content throughout the cell cycle were carried out using double fluorescent staining and cytofluorometry. Poly(ADP-ribose) synthetase content as measured immunologically was found to increase from G1 to S and G2 + M phases. Quiescent cells showed a lower content as measured immunologically of poly(ADP-ribose) synthetase than cells in the G1 phase. In exponentially growing cells, the ratio between enzyme activity of poly(ADP-ribose) synthetase over the amount of enzyme measured immunologically was found to be higher in the G2 + M phase. These results show that a cell-cycle specific event activates poly(ADP-ribose) synthetase in the G2 + M phase.

Animals↗
PubMed1988

Early events in murine erythroleukemia cells induced to differentiate. Accumulation and gene expression of the transformation-associated cellular protein p53.

Oncogenes may play a crucial role in the genetic program of cellular differentiation; even, probably, at a very early stage in this program, which can be described as pre-commitment. We have investigated the variation in, and the control level of, the accumulation of the transformation-associated cellular protein p53 in murine erythroleukemia cells induced to differentiate by hexamethylene bisacetamide. Using flow cytofluorimetry after double staining of the cells, we have found that p53 decreased from two hours after the input of the inducer, to reach a basal level of about 30% of the starting value. The stability of the protein was found to be affected neither by the inducer nor by the position of the cells in the cell cycle. Looking for the regulation mechanism of the p53 decay, we found that the mRNA started to decrease as early as half an hour after the hexamethylene bisacetamide was put in the culture medium, and that the transcription rate of the gene itself could not account for the observed down-regulation of the mRNA, suggesting a post-transcriptional control for the mRNA accumulation. This control did not require the de-novo synthesis of a protein component, as shown by cycloheximide experiments, but seemed to be governed by the induced synthesis of an RNA molecule. Hypothetical models for such a regulation process are discussed in the light of recent reports on the metabolism of mRNA.

Acetamides↗