Protein kinase CK2 in health and disease: CK2: a key player in cancer biology.

Elevated levels of protein kinase CK2 (formerly casein kinase 2 or II) have long been associated with increased cell growth and proliferation both in normal and cancer cells. The ability of CK2 to also act as a potent suppressor of apoptosis offers an important link to its involvement in cancer since deregulation of both cell proliferation and apoptosis are among the key features of cancer cell biology. Dysregulated CK2 may impact both of these processes in cancer cells. All cancers that have been examined show increased CK2 expression, which may also relate to prognosis. The extensive involvement of CK2 in cancer derives from its impact on diverse molecular pathways controlling cell proliferation and cell death. Downregulation of CK2 by various approaches results in induction of apoptosis in cultured cell and xenograft cancer models suggesting its potential as a therapeutic target.

Genomic organization and promoter characterization of the rat cyclin B1 gene.

Cyclin B1 is a key regulatory protein involved in cellular mitosis. We have cloned 1.8kb of DNA sequence upstream of the rat cyclin B1 gene translation start site from Rattus norvegicus liver genomic DNA and a commercial rat testis genomic library. The mRNA transcription start point (tsp) was determined by primer extension and mRNA end ligation followed by RT-PCR across the ligated 3' and 5' ends. An authentic tsp was confirmed approximately 100bp upstream of the translation start site. A second potential tsp was also detected approximately 32bp downstream from the first. RT-PCR analysis of rat liver poly(A)(+) RNA using 5'-derived oligonucleotide primers indicated that the 5' end sequence was present in both the 1.6 and 2. 4kb rat liver cyclin B1 mRNA species. Like many other cyclin promoters, there was no apparent TATA box upstream of the transcription initiation sites. However, computer analysis of the promoter region identified a group of consensus transcription factor binding sites, some of which are also reported in other cyclin promoters. These include those for p53, p21, Ap-1, Ap-2, Ets-1, CAATT, E-Box and Yi. We also performed luciferase reporter assays using a set of promoter deletion constructs in human HuH-7 hepatoma and HeLa carcinoma cell lines. Our results suggest that an E-Box and/or CCAAT binding sites are important for transcription, and that there may be negative regulatory elements present between 1800 and 1100bp upstream of the translation start site.

Posttranscriptional regulation of cyclin B messenger RNA expression in the regenerating rat liver.

The growing family of cyclin genes and their products have been identified as important regulatory participants in the eukaryotic cell cycle. Cyclin proteins are currently postulated to act at the G1 restriction point, entry and exit of S phase, and the G2-M transition. We have cloned a rat cyclin B complementary DNA (cDNA) and have investigated cyclin B mRNA expression and regulation in the regenerating rat liver following 70% partial hepatectomy (PH). Sequence analysis of the rat cyclin cDNA revealed greater than 82% identity to type B1 human and murine cyclin genes. The rat cyclin cDNA was used to probe Northern blots of polyadenylated enriched RNA from regenerating rat liver from 0 through 96 h post-PH. Two species of rat cyclin B transcript were detected which mapped at 1.6 and 2.4 kilobases in length. Steady-state transcript levels began to appear around 24 h post-PH, which coincides with peak DNA synthesis. However, expression of the cyclin B transcripts peaked at 48 h and was 20-fold greater than at 24 h post-PH. Smaller peaks of expression occurred at 30 and 72 h. Run-off transcription assays using nuclei isolated at various times post-PH indicated no change in transcriptional rate during the period of regeneration. In vivo mRNA half-life determinations were performed at 24, 40, and 48 h post-PH. The half-lives of both transcript species were almost identical and were determined to be greater than 12 h at 24 h post-PH, and 2.4 h at 40 and 48 h post-PH. Protein inhibition with cycloheximide increased the signal intensity of both transcripts between 48 and 54 h post-PH but had no detectable effect on 0 h transcript expression. Steady-state levels of thymidine kinase mRNA showed a similar pattern of expression by Northern analysis through 96 h post-PH as cyclin B. The present study indicates that the appearance of cyclin B mRNA in the regenerating rat liver is coincident with peak DNA synthesis, although its own peak expression is significantly delayed. Steady-state transcript levels appear to be regulated primarily by posttranscriptional events of which changes in mRNA stability may be an important determinant. We propose that the involvement of cyclin B in the cell cycle machinery is controlled at several different levels of gene expression.

Alterations in mRNA stability during rat liver regeneration.

We examined the in vivo transcriptional and posttranscriptional regulation of various genes involved in hepatocyte growth and replication that exhibited changes in steady-state mRNA levels after 70% partial hepatectomy (PH). Of the 19 genes examined by nuclear run-on assay, 17 demonstrated no change in transcriptional activity through the first 96 h of regeneration. However, results from in vivo half-life determinations indicated that changes in mRNA stability played a critical role in regulating transcript levels during liver regeneration. For many of the genes, alterations in transcript abundance correlated with similar changes in mRNA half-lives. Inhibition of protein synthesis by cycloheximide was generally associated with increased levels of mRNA expression, but no detectable changes in transcriptional rates in both control and regenerating rat liver. Finally, genomic methylation status was investigated by Southern analysis for several genes that displayed changes in mRNA stability. Interestingly, increases in mRNA half-lives for the genes p53, c-myc, H-ras, and ornithine decarboxylase were associated with decreased genomic methylation. In conclusion, regulation of gene expression beyond the immediate early phase of te cell cycle during rat liver regeneration after PH occurs predominantly at the posttranscriptional level. mRNA stability appears to be a significant factor in this control, and may itself be modulated by the methylation status of the corresponding genomic DNA.

Differential regulation of cyclin B1 RNA and protein expression during hepatocyte growth in vivo.

Cyclin genes and their products are important regulatory participants in the eukaryotic cell cycle. It is well established that cyclin B1 protein forms a complex with cyclin-dependent kinase 1 (CDK1), which, when activated, initiates mitosis. We have previously established that cyclin B1 gene expression is posttranscriptionally regulated in regenerating rat liver after 70% partial hepatectomy (PH). We now report further characterization of cyclin B1 gene expression, as well as that of CDK1 and cdc25B, in this unique in vivo model of cell proliferation. Cyclin B1 transcripts were detected by RNase protection through 96 h of liver regeneration and exhibited dramatic changes in steady-state levels. Peak expressions occurred at 24-30 h, more significantly at 42-48, and at 72 h. By Northern blot analysis, single transcripts for CDK1 and cdc25B were detected, and the temporal expression of both transcripts during liver regeneration mirrored that of cyclin B1. By Western blot and immunohistochemical analyses, cyclin B1 protein levels did not change significantly in either nuclear or cytoplasmic fractions, whereas CDK1 protein levels paralleled their associated RNA expression. Cdc25B protein levels steadily decreased from 0 to 96 h after PH. In addition, cytoplasmic protein levels of cyclin B1 exhibited a constant distribution in subfractions of microsome- and polysome-associated and free proteins. Cyclin B1 RNA also localized to these three cytoplasmic subfractions. Finally, the apparent translational activity of cyclin B1 transcripts was very similar at both 24 and 48 h after PH, in contrast to their respective mRNA half-lives. In a peroxisome proliferation model of hepatocyte growth and apoptosis, cyclin B1 and CDK1 proteins were induced in the absence of transcript up-regulation. Our results demonstrate that cyclin B1 mRNA steady-state levels are regulated posttranscriptionally in regenerating rat liver. Furthermore, the pattern of cyclin B1 transcript expression is paralleled by that of the CDK1 gene, whereas their respective protein steady-state levels provide a striking contrast. Finally, cyclin B1 is differentially regulated by an uncoupling of transcript abundance and translational processing in two in vivo models of hepatocyte growth. The abundance of cyclin B1 protein in nonreplicating cells suggests that cyclin B1 may be available for other cellular pathways in the hepatocyte.

The RNP protein, RNPS1, associates with specific isoforms of the p34cdc2-related PITSLRE protein kinase in vivo.

The PITSLRE protein kinases are members of the p34cdc2 superfamily, with >20 different isoforms expressed from two linked genes in humans. PITSLRE homologues have been identified in mouse, chicken, Drosophila, Xenopus, and possibly Plasmodium falciparum, suggesting that their function may be well conserved. A possible role for a caspase processed PITSLRE isoform has been suggested by studies of Fas- and TNF-induced cell death. However, the function of these kinases in proliferating cells is still unknown. Here we demonstrate that the 110 kDa PITSLRE isoforms (p110) are localized to both the nucleoplasm and nuclear speckles, and that these isoforms specifically interact in vitro and in vivo with the RNA-binding protein RNPS1. RNPS1 is also localized to nuclear speckles, and its over expression disrupts normal nuclear speckle organization by causing the aggregation of many nuclear speckles into approximately 6 'mega' speckles. This type of nuclear speckle aggregation closely resembles what occurs when cells are treated with several transcriptional inhibitors. These data indicate that the PITSLRE p110 isoforms interact with RNPS1 in vivo, and that these proteins may in turn influence some aspect of transcriptional and/or splicing regulation.

Modulation of apoptosis-associated genes bcl-2, bcl-x, and bax during rat liver regeneration.

Liver regeneration (LR) after 70% partial hepatectomy (PH) represents a unique in vivo model of cell cycle and gene regulation. This study was conducted to characterize apoptosis-associated gene expression during LR. The results indicated that transcripts for both bcl-x and bcl-2 exhibited similar patterns of expression during LR with peaks at 6 h post-PH. In contrast, the major 1.1-kb bax transcript exhibited peaks at 18 (P < 0.05) and 72 h (P < 0.001) post-PH. Nuclear run-on analyses for all three genes indicated no detectable transcription rate changes during LR. At 6 h post-PH, when bcl-x mRNA levels were increased by 25-fold (P < 0.001), bcl-x mRNA half-life was elevated 4-fold (P < 0.001). Similarly, bax transcript half-life increased from 2.8 h at 0 h to 4.3 h at 24 h (P < 0.001) and > 8 h at 40 h (P < 0.001) post-PH, coincident with increases in steady-state levels of mRNA. Western blot analyses of Bcl-2 and Bcl-x proteins showed no significant change through 96 h of LR, whereas Bax protein levels cycled in parallel with its mRNA. Interestingly, novel Bax- and Bcl-2-cross-reactive proteins of 31 and 32 kDa, respectively, were detected in nuclei isolated from quiescent liver. When liver growth was induced by the peroxisome proliferator clofibrate, transcript and protein levels were coupled for bcl-x but not for bax. In conclusion, the apoptosis-associated genes bcl-2, bcl-x and bax are modulated at the transcript and protein levels during LR, suggesting a role for these gene products in normal liver growth. The alterations in transcript levels occur posttranscriptionally and involve changes in mRNA stability. Furthermore, unlike bax, steady-state protein and transcript levels are uncoupled for both bcl-2 and bcl-x, suggesting a role for translational regulation during LR after PH.