Alterations in the PITSLRE protein kinase gene complex on chromosome 1p36 in childhood neuroblastoma.

p58cdc2L1, a protein kinase implicated in apoptotic signaling, is one of eight separate kinases encoded by three tandemly duplicated and linked genes, which we have termed PITSLRE A, B and C. One allele of this complex on chromosome 1 was either deleted or translocated in each of 18 neuroblastoma cell lines with cytogenetically apparent 1p alterations. A protein encoded by this locus, PITSLRE gamma 1, was absent in three of the lines and a smaller, apparently truncated, PITSLRE polypeptide was found in another line. These findings identify a novel gene complex on chromosome 1 that encodes a protein kinase subfamily. We suggest that the PITSLRE locus may harbour one or more tumour suppressor genes affected by chromosome 1p36 modifications in neuroblastoma.

Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN.

Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.

Identification of an ataxia telangiectasia-mutated protein mediated surveillance system to regulate Bcl-2 overexpression.

Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.

PITSLRE protein kinase activity is associated with apoptosis.

Minimal ectopic expression of a 58-kDa protein kinase (PITSLRE beta 1), distantly related to members of the cdc2 gene family, induces telophase delay, abnormal chromosome segregation, and decreased growth rates in Chinese hamster ovary cells. Here we show that this decrease in cell growth rate is due to apoptosis. Apoptosis is also induced by ectopic expression of an amino-terminal deletion mutant containing the catalytic and C-terminal domains of PITSLRE beta 1 but not by other mutants lacking histone H1 kinase activity or by other members of the cdc2 gene family. However, unlike the wild-type PITSLRE beta 1 over-expressors, ectopic expression of the N-terminal PITSLRE beta 1 mutant does not result in telophase delay or abnormal chromosome segregation. These results suggested that the function of this protein kinase could be linked to apoptotic signaling. To test this hypothesis, we examined levels of PITSLRE mRNA, steady-state protein, and enzyme activity in human T cells undergoing apoptosis after activation with the anti-Fas monoclonal antibody (MAb). All were substantially elevated shortly after Fas MAb treatment. In addition to new transcription and translation, proteolysis contributed to the increased steady-state levels of a novel 50-kDa PITSLRE protein, as suggested by the diminution of larger PITSLRE isoforms observed in the same cells. Indeed, treatment of the Fas-activated T cells with a serine protease inhibitor prevented apoptotic death and led to the accumulation of larger, less active PITSLRE kinase isoforms but not the enzymatically active 50-kDa PITSLRE isoform. Finally, induction of apoptosis by glucocorticoids in the same cell line, as well as by Fas MAb treatment of another T-cell line, led to a similar induction of 50-kDa PITSLRE protein levels over time. These findings suggest that (i) PITSLRE kinase(s) may lie within apoptotic signaling pathway(s), (ii) serine protease activation may be an early event in Fas-activated apoptosis of human T cells, and (iii) some PITSLRE kinase isoforms may be targets of apoptotic proteases.

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase.

Recently, we cloned two highly related human genes, hChlR1 ( DDX11 ) and hChlR2 ( DDX12 ), which appear to be homologs of the Saccharomyces cerevisiae CHL1 gene. Nucleotide sequence analysis suggests that these genes encode new members of the DEAH family of DNA helicases. While the enzymatic activity of CHL1 has not been characterized, the protein is required for the maintenance of high fidelity chromosome segregation in yeast. Here we report that the hChlR1 protein is a novel human DNA helicase. We have expressed and purified hChlR1 using a baculovirus system and analyzed its enzymatic activity. The recombinant hChlR1 protein possesses both ATPase and DNA helicase activities that are strictly dependent on DNA, divalent cations and ATP. These activities are abolished by a single amino acid substitution in the ATP-binding domain. The hChlR1 protein can unwind both DNA/DNA and RNA/DNA substrates. It has a preference for movement in the 5'-->3' direction on short single-stranded DNA templates. However, unlike other DNA helicases, the hChlR1 DNA helicase can translocate along single-stranded DNA in both directions when substrates have a very long single-stranded DNA region. The enzymatic activities of hChlR1 suggest that DNA helicases are required for maintaining the fidelity of chromosome segregation.

Disruption of the cyclin D/cyclin-dependent kinase/INK4/retinoblastoma protein regulatory pathway in human neuroblastoma.

The p16INK4a (MTS1) and pl8INK4c gene products are normal, and highly expressed, in human neuroblastoma cell lines. The retinoblastoma protein (pRb) was, nonetheless, phosphorylated and functional in these cells. Such high levels of p16INK4a/p18INK4c should normally inhibit cyclin-dependent kinase (CDK) 4 and 6 activities in cells containing functional pRb, delaying cell cycle progression and growth. These neuroblastoma cell lines express both CDK4 and CDK6 mRNA and protein, but only significant CDK6 protein kinase activity was detected in this study. In addition, CDK6 was not present in p16INK4a immune complexes in cells with significant kinase activity, although p16INK4a levels were high. Others have shown that a specific mutation in the NH2-terminal region of the CDK4 gene product can disrupt p16INK4a binding, thereby bypassing its inhibitory activity. To determine whether mutation of the CDK6 gene, or some other mechanism, is responsible for the CDK6 kinase activity in these cell lines, several complementary analyses were performed. The CDK6 gene from each cell line was examined for mutations that might affect p16INK4a binding, whereas p16INKa add-back experiments were performed with CDK6 immune complexes to assess p16INK4a function. A bona fide CDK6 mutation that disrupts p16INK4a binding and prevents inhibition of CDK6 protein kinase activity was identified in 1 of 17 neuroblastoma cell lines. The mechanism(s) responsible for disruption of p16INK4a inhibitory activity in the remaining cell lines is unknown, but these results suggest that neuroblastoma cells may bypass the cell cycle block imposed by constitutive expression of wild-type p16INK4a in novel ways.

Alternatively spliced cyclin C mRNA is widely expressed, cell cycle regulated, and encodes a truncated cyclin box.

The cyclin C protein has recently been shown to associate with a unique cyclin dependent protein kinase (cdk8) and it has been proposed that this complex may regulate RNA transcription during the cell cycle. In addition, the human cyclin C gene has been localized to human chromosome 6q21 and it was found to be frequently deleted in a subset of acute lymphoblastic leukemias (ALL's). Screening of an avian T-cell cDNA library resulted in the isolation of a cyclin C homologue as well as an abundant, yet distinct, cyclin C-related cDNA. The predicted open reading frame (ORF) of the cyclin C cDNA predicted a 283 amino acid protein that was > 99% identical to the human protein and 72% identical to the Drosophila melanogaster protein. However, the predicted ORF of the cyclin C-related cDNA predicted a much smaller 105 amino acid protein that was identical to cyclin C well into the cyclin-box region (amino acid residue 98), where it abruptly diverges and then terminates. Using PCR analysis of cDNA derived from a range of cell lines and tissues, alternative splicing of the avian cyclin C gene has been demonstrated. Furthermore, a smaller approximately 19 kDa protein that co-migrates with the in vitro transcribed and translated truncated cyclin C protein was detected in normal and virally-transformed avian cells with a cyclin C-specific antibody. Expression of alternatively spliced cyclin C mRNA and protein is regulated in a cell cycle-dependent manner reminiscent of cyclin B2. The function of this truncated cyclin C protein is not known, but its expression in avian cells suggest that this truncated cyclin C protein may participate as an early endogenously encoded cyclin C inhibitor.

Fas activates NF-kappaB and induces apoptosis in T-cell lines by signaling pathways distinct from those induced by TNF-alpha.

The p55 tumor necrosis factor (TNF) receptor and the Fas (CD95/APO-1) receptor share an intracellular domain necessary to induce apoptosis, suggesting they utilize common signaling pathways. To define pathways triggered by Fas and TNF-alpha we utilized human CEM-C7 T-cells. As expected, stimulation of either receptor induced apoptosis and TNF-alpha-induced signaling included the activation of NF-kappaB. Surprisingly, Fas-induced signaling also triggered the activation of NF-kappaB in T cells, yet the kinetics of NF-kappaB induction by Fas was markedly delayed. NF-kappaB activation by both pathways was persistent and due to the sequential degradation of IkappaB-alpha and IkappaB-beta. However, the kinetics of IkappaB degradation were different and there were differential effects of protease inhibitors and antioxidants on NF-kappaB activation. Signaling pathways leading to activation of apoptosis were similarly separable and were also independent of NF-kappaB activation. Thus, the Fas and TNF receptors utilize distinct signal transduction pathways in T-cells to induce NF-kappaB and apoptosis.

Aggressive childhood neuroblastomas do not express caspase-8: an important component of programmed cell death.

Neuroblastomas that overexpress N-Myc due to amplification of the MYCN oncogene are aggressive tumors that become very resistant to treatment by chemotherapy and irradiation. to identify tumor suppressor genes in this group of neuroblastomas we analyzed the expression and function of both apoptosis-related cell cycle regulatory genes in cell lines and patient tumor samples. We found that in a high percentage of neuroblastoma cell lines and patient samples with amplified MYCN, caspase-8 mRNA is not expressed. The caspase-8 gene, CASP8, was deleted or silenced by methylation in the neuroblastoma cell lines while methylation of its promoter region was the predominant mechanism for its inactivation in the patient tumor samples. Reintroduction of caspase-8 into the neuroblastoma cell lines resensitized these cells to drug-induced and survival factor dependent apoptosis. Subsequently others have also shown that caspase-8 is silenced by methylation in neuroblastoma and peripheral neural ectodermal tumors, and that the caspase-9 regulator Apaf-1 is silenced by methylation in melanoma cell lines and patient samples. We conclude that caspase-8 acts as a tumor suppressor gene in neuroblastomas, that its silencing provides a permissive environment for MYCN gene amplification once the tumors are treated with chemotherapeutic drugs/irradiation, and that expression of this gene in these tumor cells may be of clinical benefit. We also discuss the possible significance of the neural crest cell progenitor cell origin and the silencing of important apoptotic regulators via methylation in both neuroblastoma and melanoma tumors.

Structure and expression of chicken protein kinase PITSLRE-encoding genes.

The human PITSLRE protein kinases (PK), members of the p34cdc2 kinase family named according to the single amino acid (aa) code of an important (PSTAIRE) regulatory region [Meyerson et al., EMBO J. 11 (1992) 2909-2917], are candidate tumor suppressor gene(s) localized to human chromosome 1p36.2 and a syntenic region of mouse chromosome 4 [Lahti et al., Nature Genet. 7 (1994) 370-375; Mock et al., Mammal. Genome 5 (1994) 191-192]. At least ten isoforms of this PK family are expressed from three duplicated and tandemly linked genes in humans [Xiang et al., J. Biol. Chem. 269 (1994) 15786-15794]. We have now isolated two different species of PITSLRE PK cDNAs from chicken that encode identical polypeptides, but are clearly expressed from different genes, based on nucleotide (nt) differences. Isolation of one of the corresponding chicken PITSLRE PK genes confirms that only one of the two species of PITSLRE mRNA is expressed from this gene. Comparison of the predicted avian PITSLRE PK aa sequence to human and mouse sequences shows a high degree of sequence identity (> 91%). Like humans, the PITSLRE PK genes in chickens must be closely linked, based on fluorescent in situ hybridization (FISH) localization of these genes to a single chicken microchromosome. PITSLRE PK mRNAs are expressed in two avian B- and T-cell lines. These results suggest that the PITSLRE PK gene family has been well conserved evolutionarily, that the gene duplication observed in humans is not a recent event, and that expression of redundant PITSLRE mRNAs is observed in different vertebrate species.

Abnormalities in the p34cdc2-related PITSLRE protein kinase gene complex (CDC2L) on chromosome band 1p36 in melanoma.

The two genes encoding the PITSLRE protein kinase isoforms, CDC2L1 and CDC2L2, are localized to human chromosome band 1p36. The PITSLRE protein kinases are a part of the p34cdc2 supergene family. Several protein products of the CDC2L locus may be effector(s) in apoptotic signaling. The larger PITSLRE p110 isoforms appear to regulate some aspect of RNA splicing/transcription during the cell cycle. One or more of these genes may function as tumor suppressor genes in melanoma. Using fluorescence in situ hybridization, one allele of the CDC2L gene complex on chromosome 1 was either deleted or translocated in 8 of 14 different melanoma cell lines. We also observed mutations in the 5' promoter region of the CDC2L1 gene in four different cell lines relative to normal melanocytes using PCR-SSCP analysis and direct DNA sequencing. Western blot analysis revealed decreased level of PITSLRE protein expression in several cell lines, as well as in four surgical malignant melanoma specimens relative to normal melanocytes. Thus, the decreased PITSLRE protein expression appears to result from deletion of the CDC2L alleles and possibly by mutations within the 5' promoter region. We propose that aberrations in the CDC2L genes may contribute to the pathogenesis or progression of melanoma.

CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Use of gene knockouts in cultured cells to study apoptosis.

The avian DT40 cell system represents a novel method to generate loss of function mutations in vertebrate cells. These chicken B lymphoma cells undergo homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockout cells can then be used to determine how the cell death process is modulated after induction of apoptosis and to order components in cell death pathways. The system can be further modified, using tetracycline-responsive promoters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression constructs containing mutations or deletions in the cDNA encoding the absent protein can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal transduction pathway could also be transfected into the knockout cell line, and the resultant cells could be assayed for complementation and/or rescue of the apoptotic alteration/defect. Finally, the system has recently been adapted to allow disruption of human genes in DT40/human hybrid cell lines thereby potentially extending this system for use in studying human genes.

Structure and expression of the human p58clk-1 protein kinase chromosomal gene.

A cDNA corresponding to a 58-kDa cell division control-related protein kinase, p58clk-1, has previously been isolated, sequenced, and assigned to human chromosome 1p36. Aberrant expression of this protein kinase negatively regulates normal cellular growth. The p58clk-1 protein contains a central domain of 299 amino acids that is 46% identical to human p34cdc2, the master mitotic protein kinase. Deletion of 1p36 has been correlated to numerous tumors, and this chromosome region has been suggested to harbor a putative tumor suppressor gene on the basis of the growth characteristics of these tumors. In this report we detail the complete structure of the p58clk-1 chromosomal gene, including its putative promoter region, transcriptional start sites, exonic sequences, and intron/exon boundary sequences. The gene is 10 kb in size and contains 12 exons and 11 introns. Interestingly, the rather large 2.0-kb 3' untranslated region is interrupted by an intron that separates a region containing numerous AUUUA destabilization motifs from the coding region. Furthermore, we detail the expression of this gene in normal human tissues as well as several human tumor cell samples and lines. The origin of multiple human transcripts from the same chromosomal gene, and the possible differential stability of these various transcripts, is discussed with regard to the transcriptional and post-transcriptional regulation of this gene. This is the first report of the chromosomal gene structure of a member of the p34cdc2 supergene family.

Characterization of putative human homologues of the yeast chromosome transmission fidelity gene, CHL1.

Helicases are components of numerous protein complexes, including those regulating transcription, translation, DNA replication and repair, splicing, and mitotic chromosome transmission. Helicases unwind double-stranded DNA and RNA homo- and hetero-duplexes. The yeast CHL1 helicase has been linked to maintenance of the high fidelity of chromosome transmission during mitosis. Mutations in this gene result in a 200-fold increase in the rate of aberrant chromosome segregation with a concomitant delay in the cell cycle at G2-M, suggesting that CHL1 is required for the maintenance of proper chromosome transmission. Two highly related human cDNA clones encoding proteins which are homologous to the yeast CHL1 gene product have been isolated. Here we show that these two distinct human CHL1-related mRNAs and proteins (hCHLR1 and hCHLR2) are expressed only in proliferating human cell lines. Quiescent normal human fibroblasts stimulated to re-enter the cell cycle by addition of serum begin to express the CHL1-related proteins as the cells enter S phase, concomitant with the expression of proliferating cell nuclear antigen. Furthermore, expression of the CHL1-related mRNAs is lost when human K562 cells cease to proliferate and terminally differentiate in response to phorbol ester treatments. Human hCHLR expression is not extinguished during hemin-induced differentiation of the same cell line, which produces erythrocyte-like cells that continue to proliferate. These experiments are consistent with the requirement of this putative helicase during either S or G2-M phase but not G1. In vitro transcribed and translated hCHLR1 protein binds to both single- and double-stranded DNA, supporting the possibility that these proteins are DNA helicases. Finally, affinity-purified hCHLR1 antisera was used to demonstrate the localization of the hCHLR proteins to the nucleolus by indirect immunofluorescence as well as by cell fractionation.

Elimination of cyclin D1 in vertebrate cells leads to an altered cell cycle phenotype, which is rescued by overexpression of murine cyclins D1, D2, or D3 but not by a mutant cyclin D1.

DT40 lymphoma B-cells normally express cyclins D1 and D2 but not D3. When cyclin D1 expression was extinguished in these cells by gene knockout, specific alterations in their ability to transit the cell cycle were observed. These changes are exemplified by a delay of approximately 2 h in their progression through a normal 14-h cell cycle. This delay results in an increase in the number of cells in the G2/M phase population, most likely due to triggering of checkpoints in G2/M, inability to enter G1 normally, and/or alterations of crucial event(s) in early G1. The defect(s) in the cell cycle of these D1 "knockout" cells can be rescued by overexpression of any normal mouse D-type cyclin but not by a mutant mouse cyclin D1 protein that lacks the LXCXE motif at its amino terminus. These data suggest that the cell cycle alterations observed in the D1-/- cells are a direct effect of the absence of the cyclin D1 protein and support the hypothesis that the D-type cyclins have separate, but overlapping, functions. Elimination of cyclin D1 also resulted in enhanced sensitivity to radiation, resulting in a significant increase in apoptotic cells. Expression of any normal murine D-type cyclin in the D1-/- cells reversed this phenotype. Intriguingly, expression of the mutant cyclin D1 in the D1 -/- cells partially restored resistance to radiation-induced apoptosis. Thus, there may be distinct differences in cyclin D1 complexes and/or its target(s) in proliferating and apoptotic DT40 lymphoma B-cells.

2-Aminopurine overrides a late telophase delay created by ectopic expression of the PITSLRE beta 1 protein kinase.

Minimal overexpression of the PITSLRE beta 1 protein kinase in CHO cells leads to a marked delay in late mitosis. We have previously shown that this delay is characterized by the presence of substantially increased numbers of tubulin midbodies, inhibition of cytokinesis, and numerous multinucleated and micronucleated cells. Others have shown that the protein kinase inhibitor 2-aminopurine (2-AP) is capable of overriding drug induced cell cycle blocks. In this study we demonstrate that the late mitotic delay and altered cellular morphology caused by ectopic expression of the PITSLRE beta 1 protein kinase can be overcome by 2-aminopurine treatment. Furthermore, 2-aminopurine inhibits PITSLRE beta 1 protein kinase activity in vivo, but does not effect p34cdc2 protein kinase activity in a similar manner.

Laser light suicide of proliferating virus-specific CD8+ T cells in an in vivo response.

Virus-specific CD8+ CTL precursor (CTLp) frequencies are a function of rates of clonal expansion and loss, both of which are difficult to assess in vivo. Proliferating T cells incorporate the nucleoside analogue 5-bromo-2'-deoxyuridine (BrdU), making them sensitive to light-induced apoptotic cell death (suicide). Mice were infected with an influenza A virus, then given 50 mg of BrdU 24 h before sampling. Exposure of freshly isolated, CD8-labeled T cells to the laser beam of the flow cytometer resulted in the elimination of > 90% of the responding CTLp. The effect was obvious for the regional lymph node from day 6 to day 12 after priming, indicating continued cycling over a 7-day interval. Thus, as CTLp frequencies remain fairly constant from 7 to 30 days after infection, the persistent increase (> 30x) in numbers must be accompanied by a very substantial loss of virus-specific CD8+ T cells.