Control of eukaryotic cell cycle progression by phosphorylation of cyclin-dependent kinases.
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Animals↗
Biomedical subjects
J Nourse
Publications and source records attributed to J Nourse.
Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle.
Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen independence occurs in the mid-to late G1 phase of the cell cycle and is called the restriction point. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulated the cyclin-dependent kinase (CDK) inhibitor p27Kip1. This was correlated with inactivation of essential G1 cyclin-CDK complexes and with cell cycle arrest in G1. The ability of specific mitogens to allow transit through the restriction point paralleled their ability to down-regulate p27, and antisense inhibition of p27 expression prevented cell cycle arrest in response to mitogen depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic signals to the cell cycle at the restriction point.
3T3 Cells↗
The mechanism of action of cyclosporin A and FK506.
The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin suppress the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA, FK506, and rapamycin bind to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin and FK506/FKBP the target is calcineurin. Because of the large surface area of interaction of the drug-immunophilin complex with calcineurin, FK506 and CsA have a specificity for their biologic targets that is equivalent to growth factor-receptor interactions. To date, all the therapeutic as well as toxic effects of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the cytosolic component of the nuclear factor of activated T cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. The purpose of this article is to illustrate the means by which these drugs produce immunosuppression.
Animals↗
Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.
The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.
Animals↗
PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1.
Two new homeobox genes, PBX2 and PBX3, were isolated on the basis of their extensive homology to PBX1, a novel human homeobox gene involved in t(1;19) translocation in acute pre-B-cell leukemias. The predicted Pbx2 and Pbx3 proteins are 92 and 94% identical to Pbx1 over a large region of 266 amino acids within and flanking their homeodomains, but all three proteins diverge significantly near their amino and carboxy termini. Chromosome in situ hybridizations demonstrated that the PBX genes are not clustered but map to separate chromosomal loci: PBX1, 1q23; PBX2, 3q22-23; PBX3, 9q33-34. Expression of PBX2 or PBX3 was not restricted to particular states of differentiation or development, as mRNA transcripts of these genes were detected in most fetal and adult tissues and all cell lines, unlike PBX1, which is not expressed in lymphoid cell lines. Similar to PBX1 RNA, PBX3 RNA is alternatively spliced to yield two translation products with different carboxy termini, a feature not observed for PBX2. Their extensive sequence similarity and widespread expression suggest a generalized, overlapping role for Pbx proteins in most cell types. Differences in their amino and carboxy termini may modulate their activities, mediated in part by differential splicing and, for PBX1, protein fusion following t(1;19) chromosomal translocation.
Amino Acid Sequence↗
Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor.
The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.
Amino Acid Sequence↗
Molecular analysis of the t(1;19) breakpoint cluster region in pre-B cell acute lymphoblastic leukemias.
The t(1;19) chromosomal translocation in acute lymphoblastic pre-B cell leukemias involves the gene E2A for helix-loop-helix (HLH) proteins E12 and E47, ubiquitous transcriptional proteins implicated in the regulation of various lymphoid and nonlymphoid genes. To characterize the molecular features of the t(1;19)(q23;p13) translocation, we molecularly cloned breakpoint DNA from t(1;19)-carrying pre-B cell leukemias. In all cases, breakpoints on chromosome 19 occurred within 2 kb of each other in a single intron of the E2A gene. This clustered arrangement resulted in specific truncation of the E2A gene and transcript, with loss of sequences encoding the basic DNA-binding and HLH dimerization motifs from the derivative 19 chromosome. In contrast, breakpoints on chromosome 1 were distributed over a large region and could not be linked to exonic sequences of the PBX1 gene, although identical chromosome 1 sequences are joined to E2A sequences in 1;19 fusion transcripts. These data show that the 1;19 translocation consistently results in exchange of 3' exons encoding the HLH motifs of E2A with DNA from chromosome 1 to form a fusion gene on the derivative 19 chromosome.
Blotting, Southern↗
Concurrent activation of c-myc and inactivation of bcl-2 by chromosomal translocation in a lymphoblastic lymphoma cell line.
We have characterized a chromosomal translocation in a cell line (SU-DUL5) established from a patient with lymphoblastic lymphoma in which the c-myc gene on chromosome 8 was juxtaposed to a t(14;18). Cytogenetic analysis of this cell line showed 14q+, 18q-, and 8p+q+ marker chromosomes in the absence of t(14;18). Genomic Southern blot analysis showed juxtaposition of the immunoglobulin heavy chain joining region (JH) with chromosome 18 near the minor breakpoint cluster region (mcr) of the bcl-2 gene. There was also a rearranged c-myc gene detectable with a 5' c-myc probe. Molecular cloning studies showed that the c-myc gene was joined to chromosome 18 DNA. Nucleotide sequence analysis of cloned breakpoint DNA revealed that the crossover between chromosomes 8 and 18 occurred at the 3' end of the bcl-2 gene resulting in replacement of the bcl-2 gene on the 14q+ chromosome with the c-myc gene. As a result of this translocation the SU-DUL5 cell line contains no detectable bcl-2 mRNA or protein but has abundant levels of c-myc mRNA. Our data suggest that bcl-2 inactivation occurred simultaneously with c-myc translocation in a B cell lymphoblastic lymphoma.
Adult↗
Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas.
A majority of t(14;18) translocations have been shown to cluster at one of two sites on chromosome 18, called the major breakpoint region (mbr) or the minor cluster region, (mcr), which map within or flanking the bcl-2 proto-oncogene, respectively. We have determined the nucleotide sequence for a portion of the mcr, and constructed oligonucleotides that were used to perform the polymerase chain reaction (PCR) in conjunction with universal immunoglobulin primers to specifically amplify t(14;18) breakpoints in DNA obtained from follicular lymphomas. Eight of ten breakpoints that were detectable on Southern blots using DNA probes for the mcr could be detected due to specific amplification by the PCR technique using an mcr-specific primer. Direct nucleotide sequencing of the enzymatically amplified DNAs showed that the breakpoints clustered within a 500 nucleotide region, and five occurred within three nucleotides of each other. These data show a remarkable clustering of some t(14;18) breakpoints at a site on chromosome 18, at least a 30-kb distance from the bcl-2 gene. Our findings also indicate that mcr-specific primers may be used in conjunction with previously described mbr-specific primers in a highly sensitive DNA amplification technique to detect a large fraction of t(14;18) breakpoints.
Base Sequence↗
The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation.
We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation.
Animals↗
"Those who came first".
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History of Nursing↗