Biomedical subjects

A A Nordin

Publications and source records attributed to A A Nordin.

At least 19 recordsLinked to original sources

PubMed2001

The expression and characterization of cyclin-dependent kinase 6 during the activation of murine G0 T-cells.

Recent evidence strongly suggest that the D type cyclins with cdk4 and cdk6 form holoenzymes that regulate cell cycle events earlier in G1 than cyclin E/cdk2 complexes which functions near the G1/S transition. In human T lymphocytes cdk6 has been shown to be the initial retinoblastoma protein kinase detectable at mid G1. Following activation of splenic derived murine G0T-cells, cdk6, cyclin D2 and D3 specific mRNAs were detected early in G1 and reached maximal levels prior to or near G1/S. The phosphorylation of retinoblastoma protein from T-cells was detected very early in G1 and was associated mainly with cdk6/cyclin D2 complexes which accounted for a minor portion of the total cellular cdk6 contained in the cytoplasmic fraction of T-cells and mostly in the catalytically inactive form.

Animals↗

PubMed2000

Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells.

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.

CDC2-CDC28 Kinases↗

PubMed1998

Characterization of the murine cdc2 gene.

A 1204 bp full-length cDNA encoding murine cdc2 was isolated and used as a probe to obtain four overlapping cdc2 genomic clones which span the entire cdc2 sequence. Characterization of these clones revealed that the cdc2 mRNA is distributed over 28 kb and in 8 exons ranging in size from 63 to approximately 303 bp. Since the 5'-untranslated sequence is interrupted by intron 1, the initiation codon is located in exon 2. The PSTAIRE region is in exon 3, and the stop codon is in exon 8. Primer extension analysis of cdc2 mRNA isolated from immobilized anti-CD3 activated T-cells demonstrated that the murine cdc2 gene utilizes multiple transcriptional start sites. No consensus sequence for a TATA box exists at an appropriate position within the promoter region. Instead, several putative transcription factor binding sites for PEA3, CREB, C/EBP, E box factor, YY1, ATF-like, Spl, and E2F were detected. Analysis of the promoter activity of the 5'-flanking region suggests that the sequence between -188 to -38, which possesses two Spl and one E2F motif, contains a major positive regulatory activity for the expression of murine cdc2.

3T3 Cells↗

PubMed1998

Identification of cdk2 binding sites on the p27Kip1 cyclin-dependent kinase inhibitor.

A cdk2 binding domain on p27Kip1 located within the sequence of amino acids 53-85 was further characterized by generating a series of point mutations within amino acid residues 62-75. Two regions, FDF (residues 62-64) and GXY (residues 72 and 74), were identified within the beta hairpin region of p27Kip1. Mutations within these regions essentially completely inhibited the binding to in vitro translated cdk2 and cdk2/cyclin E complexes formed in vitro or in vivo. The p27Kip1 GST-fusion protein of the point mutation that replaces phenylalanine at residue 64 to alanine (F64A) showed approximately twofold less inhibition of cdk2 kinase activity. The cellular response to the introduction of the F64A mutant form of p27Kip1 was compared to that of p27Kip1 wild type by transfecting HeLa cells with constructs of full length sense and antisense coding sequences. Overexpression of the F64A mutant form of p27Kip1 bound significantly lower levels of cdk2 as compared to wild type and did not affect the cdk2 related kinase activity of the transfected HeLa cells. Overexpression of wild type p27Kip1 resulted in a reduction of the level of cdk2 kinase activity and effectively suppressed the growth of the transfected HeLa cells.

Animals↗

PubMed1998

The differential catalytic activity of alternatively spliced cdk2 alpha and cdk2 beta in the G1/S transition and early S phase.

Progression through the G1/S transition of the cell cycle is regulated by cyclin E/cdk2 and cyclin A/cdk2 complexes. We demonstrate that there are two forms of murine cdk2 (cdk2 alpha and beta). Cdk2 alpha consist of 298 amino acids, while cdk2 beta contains a 48-amino-acid insert between Met (196) and Val (197) of cdk2 alpha. Cdk2 beta results from differential splicing of the primary RNA transcript of the cdk2 gene. Although human cdk2 genomic DNA contained the sequence of the insert for the beta form, cdk2 beta was not detected by either Western blot or RT-PCR in human T-cells or several other human cell lines. Cdk2 beta expression in murine cells was similar to that of the phosphorylated, catalytically active form of cdk2 alpha. Cdk2 alpha and cdk2 beta have very similar binding activity to cyclin E and to the cdk inhibitor p27Kip1. The alternatively spliced cdk2 beta possesses catalytic activity in vivo and in vitro. The differential catalytic activity of these two forms of cdk2 suggests that cdk2 alpha and cdk2 beta may perform different functions at or near the G1/S transition and early S phase.

Alternative Splicing↗

PubMed1997

Overexpression of cyclin E and cyclin-dependent kinase inhibitor (p27Kip1): effect on cell cycle regulation in HeLa cells.

Transfection of the epithelioid cell line HeLa with p27(Kip1) resulted in an accumulation of cells in the G1 phase of the cell cycle. Although the cellular level of cdk2 was not decreased as a result of this transfection, there was a significant decline in the cdk2 associated kinase activity. Restoration of the cdk2 kinase activity was proportional to the amount of cotransfected cyclin E plasmid DNA. Overexpression of cyclin E also reversed the p27(Kip1) mediated G1 growth arrest. These findings suggest that the overexpression of cyclin E reverses the p27(Kip1) mediated G1 growth arrest by binding the inhibitor restoring the cdk2 associated kinase activity necessary for the G1/S transition.

Cell Cycle↗

PubMed1997

The regulation of p27Kip1 expression following the polyclonal activation of murine G0 T cells.

Polyclonal activation of murine G0 T cells with immobilized anti-CD3 induces entry into the cell cycle as well as the subsequent cytokine-dependent proliferative response. G0 T cells express high levels of p27Kip1 protein and specific mRNA, which decline rapidly following activation. The decline in the expression of p27Kip1 and sequestering of the inhibitory protein by cdk4 and cdk6 correlated with the increase in cdk2 kinase activity during the G1 phase. Anti-CD3 activation of G0 T cells in the presence of cyclosporin A or rapamycin inhibited the down-regulation of p27Kip1, the cellular levels of the inhibitor remained high, and the cells remained in the G1 phase. PBu2 activation of G0 T cells also did not result in the down-regulation of p27Kip1 and the cells remained in G1. In each instance IL-2 restored the down-regulation of p27Kip1, resulting in a significant reduction in the level of the inhibitor, and stimulated the cells to progress through the cell cycle. Jurkat cells transfected with the p27GL-988 plasmid containing +1 to -988 nt of the p27Kip1 promoter region and subsequently exposed to rIL-2 resulted in a significant reduction in the activity of the p27Kip1 promoter. These findings suggest that in addition to providing the signals required for activated T cells to traverse G1/S, IL-2 also influences the promoter function of p27Kip1, which effectively induces transcriptional down-regulation of the gene.

Animals↗

PubMed1996

Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1.

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements.

Animals↗

PubMed1996

The cdk2 binding domain of p27Kip correlates with the inhibition of the kinase activity of cdk2/cyclin complexes.

The cyclin-dependent kinase inhibitor p27Kip binds to cyclin/cyclin-dependent kinases and preferentially inhibits the catalytic activity of cdk2 and cdk4. The cdk2 binding domain of p27Kip was determined using a series of N-terminal truncated and point mutations of GST-p27Kip. The binding domain was contained within amino acid residues 53-85. The inhibition of the catalytic activity of preformed functional cdk2/cyclin complexes by P27KiP was also associated with the binding domain.

Amino Acid Sequence↗

PubMed1996

Staurosporine-induced G1 arrest is associated with the induction and accumulation of cyclin-dependent kinase inhibitors.

The addition of 10 nM staurosporine (ST) to MDA 361 breast carcinoma cells induces a G1 arrest, which correlates with the loss of the catalytic activity of the G1-associated cyclin-dependent kinases (cdks) and increased levels of underphosphorylated retinoblastoma protein. This treatment resulted in a slight but detectable reduction in the protein levels of cdk6 but did not reduce the levels of cdk2, cdk4, or the D cyclins. The level of cyclin E declined initially but returned to normal levels 24 h after exposure to 10 nM ST. Because the levels of the G1 cdks and cyclins did not correlate with loss of kinase activity, the role of the cdk inhibitors involved in regulating the activity of the G1-associated cdks was investigated. The significant reduction in cdk activity observed in MDA 361 cells treated with ST for 24 h correlated with increased levels of p18 and p27Kip. The inhibition of kinase activity of preformed cdk2 complexes by lysates of MDA 361 cells that had been treated with 10 nM ST for 24 h was shown to be due to p27Kip. The reduction in the level of the active phosphorylated form of cdk2 also correlated with an increase in the level of p27Kip, which has been shown to inhibit the phosphorylation of the activating Thr-160 residue of cdk2. These results indicate that treatment of MDA 361 cells with 10 nM ST induces a significant increase in the levels of several cdk inhibitors that appear to be responsible for the observed G1 arrest.

Breast Neoplasms↗

PubMed1995

A novel cytoplasmic substrate for cdk4 and cdk6 in normal and malignant epithelial derived cells.

Cyclins and cyclin-dependent kinases (cdk) have been identified as important regulators of cell replication. Molecular alteration in the cdk pathways appear to be important in cancer with some cyclins (eg cyclin D and E) proposed to be oncogenes and some inhibitors of cdk (eg p16) proposed to be tumor suppressor genes. In human breast carcinoma cell line MDA361 both cyclin D and E are overexpressed and cdk 4 and 6 are the predominate kinases which phosphorylate retinoblastoma protein and to a greater extent a novel 88 kDa protein. This 88 kDa protein was detected as a significant substrate in five of seven breast carcinoma cell lines, three lung carcinoma cell lines as well as in primary breast and lung epithelium. Normal human and murine T lymphocytes and established lymphoid cell lines are devoid of this protein and minimal amounts were detected in normal human fibroblast. In contrast to retinoblastoma protein, the 88 kDa protein appears to be more prevalent in the cytosolic than the nuclear subfraction. The phosphorylation of this 88 kDa protein by the G1 associated cdks suggest that this protein may represent another targeted substrate regulating the G1 phase of the cell cycle.

Animals↗

PubMed1995

Anti-CD3-induced apoptosis in T-cells from young and old mice.

Light scatter measurements using flow cytometry indicated that T cells from young and old mice undergo apoptosis following activation with immobilized anti-CD3. The percentage of cells in apoptosis after 20 h activation was significantly greater (p < .001) in cultures containing cells from older animals. The mean percentages of apoptotic T cells from young and old mice after 20 h activation were 19.3% and 33.0%, respectively. The proportion of viable cells after 20 h activation was significantly higher (p < .003) in the young (mean = 78.4%) than in the old animals (mean = 65.8%). Simultaneous measurements of light scatter and fluorescence indicated that apoptotic T cells contained both the CD4+ and the CD8+ T-cell phenotypes. The frequency of apoptotic CD8+ T cells was elevated (p < .007) in older animals, where the mean percentage was 15.1%, compared to 5.3% in the young. The most dramatic difference between young and old (p < .0008) was seen in the percentages of viable CD4+ T cells after 20 h activation. The mean viable CD4+ T-cell percentage was 33.7% in- the young and 21.4% in the old. CD4+ cells expressing high levels of CD45RB (CD45RBhi) after activation for 20 h possessed light scatter and bright fluorescence properties characteristic of viable cells, whereas CD4+/CD45RBlo density cells could be identified as apoptotic based on their decreased CD4 fluorescence and scatter characteristics. CD4+ cells from young animals were predominantly CD45RBhi, whereas CD4+ cells from the old had greater levels of CD454RBlo cells. In addition to light scatter changes, measurement of DNA content after 40 h activation revealed the presence of a sub-G1 DNA apoptotic peak and a viable cell cycle distribution. After 40 h of activation, there was an increase in the percentage of apoptotic cells in both young and old mice, with the greatest increase seen in the cells from older animals. Further evidence supporting the process of apoptosis in 40 h-activated cells was confirmed by the appearance of DNA strand breaks detected by in situ nick translation.

Aging↗

PubMed1994

Up-regulation of c-myc induces the gene expression of the murine homologues of p34cdc2 and cyclin-dependent kinase-2 in T lymphocytes.

The expression and/or up-regulation of several early T cell activation genes is dependent on signals transmitted through the interaction of IL-2 and IL-2R well before entry of the cells into S phase. In these studies, murine G0 T cells activated by immobilized anti-CD3 and subsequently blocked in late G1 expressed normal surface levels and mRNA for IL-2R alpha, IL-2R beta, and transferrin receptor (TfR). However, there was no expression of p34cdc2, and cyclin-dependent kinase (cdk)-2 was not up-regulated even in the presence of exogenous rIL-2. In addition the accumulation of c-myc-specific mRNA and protein was significantly reduced. Pretreatment of G0 T cells with c-myc antisense oligonucleotide effectively reduced the level of specific c-myc protein induced by activation of the cells by immobilized anti-CD3. The presence of antisense c-myc oligonucleotide inhibited the expression of cdc2 and cdk2 without affecting the expression of IL-2R alpha and blocked the activated T cells in the G1 phase. Together these studies demonstrate that c-myc regulates the expression of these cdk and suggest a role for c-myc in the G1/S transition.

Animals↗

PubMed1993

Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in p56lck kinase activity and the tyrosine phosphorylation of a 97-kDa protein.

The addition of recombinant interleukin 2 (rIL-2) to anti-CD3-activated murine G0 phase T cells results in an increased level of tyrosine phosphorylation of a single 97-kDa protein. The degree of tyrosine phosphorylation paralleled the amount of rIL-2 added and correlated with the extent of DNA synthesis. IL-2 treatment resulted in a transient increase in p56lck kinase activity without detectable modification of its level of tyrosine phosphorylation and gel mobility. When G0 T cells were activated by phorbol dibutyrate in the absence of IL-2, the high-affinity IL-2 receptor (IL-2R) expressed failed to induce a proliferative signal, and neither the tyrosine phosphorylation of the 97-kDa protein nor the transient increase in p56lck kinase activity was detected. Northern analysis of the total RNA extracted from these cells showed the accumulation of IL-2R alpha chain-specific mRNA but neither c-myc nor cdc2 mRNA was expressed. The addition of 100 nM rIL-2 to T cells activated by phorbol dibutyrate was able to induce a proliferative response, and under these conditions tyrosine phosphorylation of the 97-kDa protein, the transient increase in p56lck kinase activity, and specific mRNA for IL-2R alpha chain, c-myc, and cdc2 were detected. Unstimulated G0 T cells responded to 100 nM rIL-2 in the same manner as phorbol dibutyrate-activated cells. Irrespective of the signal-transducing structures involved, the IL-2-induced proliferative response closely correlates with an increase in p56lck kinase activity along with the tyrosine phosphorylation of a 97-kDa protein.

Animals↗

PubMed1993

Identification and quantitation of apoptotic cells following anti-CD3 activation of murine G0 T cells.

Multiparameter flow cytometry and cell sorting were used to examine the process of apoptosis after activation of murine resting T cells with immobilized anti-CD3. Activated T cells treated with Hoechst 33342 (HO-33342) and analyzed by flow cytometry showed two major cell populations of high and low fluorescence. These populations were sorted and the DNA extracted and subjected to electrophoresis. Electrophoresis of DNA extracted from T cells showing a low level of HO-33342 fluorescence (HO-Low) resulted in a typical ladder pattern characteristic of internucleosomal DNA degradation associated with apoptosis, whereas the cellular DNA of the cells showing a high level of fluorescence (HO-High) showed a narrow high molecular weight band. Multiparameter analysis further indicated that cells with HO-High characteristics possessed corresponding high-FSC/low-SSC properties, whereas HO-Low cells formed a cluster of low-FSC/high-SSC cells. Analysis of the DNA extracted from cells sorted on the basis of scatter properties alone confirmed that the low-FSC/high-SSC population contained the apoptotic cells and that the high-FSC/low-SSC population was comprised of viable cells. This methodology allowed us to determine the percentage of apoptotic cells following anti-CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8+ cells in the cell cycle.

Animals↗

PubMed1992

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.

Amino Acid Sequence↗

PubMed1991

T cell activation in the absence of interleukin 2 (IL 2) results in the induction of high-affinity IL 2 receptor unable to transmit a proliferative signal.

Although interleukin 2 (IL 2) clearly up-regulates the expression of the p55 chain of the IL 2 receptor (IL 2R) little is known about its role in the induction of the high-affinity IL 2R. Resting T lymphocytes were induced to express IL 2R under experimental conditions in which IL 2 production was not induced or was prevented. Under these conditions high- and low-affinity IL 2R were easily demonstrated by Scatchard analysis. Northern blot analysis confirmed the accumulation of p55 specific mRNA and the absence of the IL 2 transcript. High-affinity IL 2R induced in the complete absence of IL 2 were unable to transmit a proliferative response unless exposed to extremely high concentrations of IL 2. The addition of picomolar amounts of recombinant IL 2 or the initiation of endogenous IL 2 production during the induction period restored the functionality of high-affinity IL 2R. Also, T cells induced to generate IL 2 displayed functional high-affinity IL 2R even in the presence of monoclonal antibodies blocking extracellular IL 2 and IL 2R. These results indicate that the presence of IL 2 during the early phase of T cell activation is an absolute requirement for the induction of fully operational high-affinity IL 2R and that low amounts of intracellular IL 2 are sufficient to confer functional properties to these receptors. The data also suggest that an intracellular as well as an extracellular high-affinity structure, expressed as a consequence of cell activation, is responsible for conferring competence to the high-affinity IL 2R involved in IL 2-dependent proliferation.

Animals↗

PubMed1988

Restricted expression of mitogen-induced high affinity IL-2 receptors in aging mice.

Several lines of indirect evidence suggest that the number and/or affinity of IL-2R expressed by activated T lymphocytes declines with age and that this decline is implicated in the age-related proliferative impairment of Ag or mitogen-stimulated T cells. In an attempt to provide a direct demonstration of such a defect, various experimental approaches were used to analyze the expression of high and low affinity IL-2R as well as their functional properties in relation to age in purified populations of murine T lymphocytes. IL-2R were induced by Con A-activation which involves a transmembrane signaling mechanism or by exposure to phorbol dibutyrate (PDBu) which bypasses such a pathway. Consistent with the previously reported age-related defect in signal transduction, a major deficiency in the expression of high affinity IL-2R was observed in mitogen-activated cells derived from aged animals. As expected, PDBu-induction circumvented the transmembrane signaling defect and resulted in the restoration of a measurable amount of high affinity IL-2R expressed by cells from aged mice early after activation. The functional properties of the IL-2R expressed as a consequence of Con A or PDBu induction were investigated by assessing the proliferative response induced through the high affinity IL-2R as compared to that mediated by the beta-chain alone. Although Con A-induction resulted in a decreased expression of high affinity IL-2R by T lymphocytes derived from aged mice, the ability of these receptors as well as that of their beta-chain component to transmit a proliferative signal was identical in both age groups. In contrast, PDBu induced in both cell populations the expression of functionally aberrant IL-2R, unable to signal for proliferation unless excessively high concentrations of rIL-2 were available. The quantitative minimal estimate of the frequency of Con A-activated, IL-2-responsive cells showed a fourfold age-associated decrease, confirming the inability of a subpopulation of T lymphocytes from aged mice to express a sufficient density of high affinity IL-2R as a consequence of mitogenic activation.

Aging↗