Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells.

CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2ß showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.

Modified tetrahalogenated benzimidazoles with CK2 inhibitory activity are active against human prostate cancer cells LNCaP in vitro.

A series of novel CK2 inhibitors, tetrahalogenated benzimidazoles carrying an aminoalkylamino group at position 2, has been prepared by nucleophilic substitution of the respective 2,4,5,6,7-pentabromobenzimidazoles and 2-bromo-4,5,6,7-tetraiodobenzimidazoles. The new derivatives as well as some previously obtained tetrahalogenobenzimidazoles, including 4,5,6,7-tetrabromobenzimidazole (TBI) and 4,5,6,7-tetraiodobenzimidazole (TIBI), were evaluated for activity against the hormone-sensitive human prostate cancer cell line LNCaP. The activity of 2-aminoalkylamino derivatives was notably higher (LD(50) 4.75-9.37 µM) than that of TBI and TIBI (LD(50) ≈ 20 µM). The determination of the LD(50) value identified the 2-aminoethylamino-4,5,6,7-tetraiodobenzimidazole with an additional methyl group at position 1 (6) as the most efficient compound (LD(50): 4.75 ± 1.02 µM). Interestingly, there was no clear correlation between cell viability and apoptosis induction indicating additional cell death mechanisms.

Down-regulation of CK2 activity results in a decrease in the level of cdc25C phosphatase in different prostate cancer cell lines.

Protein kinase CK2 is implicated in the regulation of the cell cycle. In addition to a variety of functions, CK2 has anti-apoptotic properties. So far the role of CK2 linking both pathways in the cell is not clear. Some years ago we found that CK2 phosphorylates cdc25C, one member of the cdc25 family of proteins. In this study, we showed that inhibition of CK2 activity by three different inhibitors led to a down-regulation of the level of cdc25C. Inhibition of CK2 activity by transfecting the dominant-negative CK2α subunit also resulted in a down-regulation of the level of cdc25C whereas inhibition of CK2α' had no effect on the cdc25C level. In both cases, we observed apoptosis by PARP cleavage as well as by an increase in γH2AX phosphorylation. These results show that down-regulation of the level of cdc25C is not a prerequisite for the induction of apoptosis.

Protein kinase CK2 and new binding partners during spermatogenesis.

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory ß-subunit and the catalytic α- or α'-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2ß and TNP1 were found in early spermatogenesis. CK2α, CK2α', TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2ß was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

Contribution of the individual subunits of protein kinase CK2 and of hPrp3p to the splicing process.

We have recently shown that protein kinase CK2 binds to the splice factor hPrp3p. Moreover, CK2 phosphorylates hPrp3p in vitro and in vivo. Here we analysed the spliceosome for the presence of individual subunits of CK2. By cell fractionation experiments we found that CK2alpha, CK2alpha', the spliceosome specific SmB/B' and hPrp3p protein co-localize in the nucleus. A sucrose density gradient analysis revealed that the individual subunits of CK2 co-sedimented at least in part with the SmB/B' protein. We further show by co-immune precipitation experiments that CK2alpha is associated with the U4/U6*U5 tri-snRNP specific protein 61K. In vitro splice assays with nuclear extracts depleted from CK2alpha, CK2alpha' and hPrp3p, respectively, revealed that CK2alpha and hPrp3p are absolutely necessary for an efficient splicing whereas CK2alpha' seems to be dispensable. Furthermore, these data support the notion about individual roles for CK2alpha and CK2alpha' in the splicing process.

Growth inhibition and apoptosis induction in ovarian cancer cells.

Standard therapy for the treatment of ovarian cancer is radical surgery followed by radiation and/or chemotherapy using cisplatin and paclitaxel. Unfortunately, some patients relapse after this first line chemotherapy and some patients become platinum-refractory. Therefore, we analyzed two different ovarian carcinoma cell lines for their sensitivity for gamma-irradiation and treatment with cisplatin, irinotecan, paclitaxel and gemcitabine. We found that both cell lines were rather resistant against gamma-irradiation and treatment with cisplatin and irinotecan whereas paclitaxel and gemcitabine resulted in a considerable reduction of the viability of the cancer cells. Both paclitaxel and gemcitabine treatment resulted in the induction of apoptosis. This sensitivity profile might be due to a particular subset of p53, which reacted with monoclonal antibodies DO-1 and PAb1801 but not with PAb1620 and PAb421. Gemcitabine and paclitaxel are highly efficient in the induction of apoptosis in ovarian cancer cells, which express a particular subset of the growth suppressor protein p53. Thus, a sensitivity profile for each ovarian carcinoma seems to be highly recommended before starting treatment.

Immunologically defined subclasses of the protein kinase CK2 beta-subunit in prostate carcinoma cell lines.

Both, the activity as well as the expression of protein kinase CK2 is enhanced in various cancer types and in established tumour cell lines. This phenomenon is not due to an increase in the CK2 message but rather to posttranscriptional and posttranslational mechanisms. In order to get an insight into these posttranslational modifications we analyzed CK2 in prostate cancer cell lines, which differ by their hormone-sensitivity. We found that the CK2 activity is significantly higher in hormone-refractory than in hormone-sensitive cells although the amount of the catalytic alpha- and alpha'- subunits is comparable. In contrast, we detected seemingly lower amounts of the regulatory beta-subunit in the hormone-refractory cell lines, which later turned out to be an immunologically defined subclass. This subclass is realized by a phosphate group, which is attached to serine 209. The phosphorylation occurs in vivo during mitosis and is executed by the p34(cdc2)/cyclin B kinase. As this phosphorylation enhances the CK2 activity this change might well account for the higher activity of CK2 in prostate cancer cells.

Detailed chromosomal characterization of the breast cancer cell line MCF7 with special focus on the expression of the serine-threonine kinase 15.

Although the cell line MCF7 is widely used in breast cancer research, its cytogenetic properties have not been thoroughly investigated so far. As conventional G-banding analysis cannot resolve the complex chromosome aberrations, we investigated MCF7 cells using molecular-cytogenetic methods, with particular attention to the DNA amplification site on chromosome 20q. With spectral karyotyping we found numerous unbalanced chromosome translocations, and with comparative genomic hybridization we detected many quantitative genomic imbalances. Furthermore, we analyzed the amplified region at 20q with the candidate tumour susceptibility gene STK15 in detail by fluorescence in situ hybridization, whole chromosome painting, immunohistochemistry, Western blot and expression analysis. In MCF7 interphase cells we found increased copy number of the STK15 gene associated with overexpression of STK15 mRNA. Accordingly, STK15 protein is overexpressed as compared to normal human fibroblasts in Western blot analysis. Overexpression of STK15 mRNA and protein is disproportionally stronger than that expected from the single additional copy of the STK15 gene. These data indicate that the highly increased level of STK15 protein in MCF7 cannot be explained by gene amplification alone. Apparently, secondary mechanisms of gene up-regulation are involved. This observation may be of general interest with regard to the activation of oncogenes in tumour cells.

Phosphorylation of mdm2 at serine 269 impairs its interaction with the retinoblastoma protein.

We previously reported that protein kinase CK2 phosphorylates the human mdm2 (hdm2) protein at serine residue 269. This phosphorylation site is located in the central acidic, highly-conserved region of mdm2, which is responsible for the interaction with a number of proteins. Studying the influence of phosphorylation of mdm2 by CK2 upon interaction with some of these binding partners, we found that the retinoblastoma (Rb) protein bound more strongly to the unphosphorylated mdm2 than to its CK2-phosphorylated counterpart. An S269 phosphospecific antibody was generated, and reacted with a 60 kDa subpopulation of mdm2 in human cells. We created a mutant mdm2 with a serine to aspartic acid exchange at position 269, which was used to transfect mdm2-/- cells. Cells transfected with the S269D mutant exhibited a different growth behavior than wild-type mdm2-expressing cells, which might be attributed to the altered Rb-mdm2 interaction.

Temperature-sensitive ovarian carcinoma cell line (OvBH-1).

OvBH-1 cells from a patient with ovarian clear cell carcinoma were established and their biochemical status was analyzed. Cells grown at 37 degrees C exhibited normal cell cycle distribution, whereas the cells shifted to 31 degrees C were arrested in the G(2) / M phase of the cell cycle. Immunochemical analysis using anti-p53 antibodies (DO-1, PAb240, PAb421, and PAb1620) revealed that only the DO-1 antibody reacted with p53 with a high and similar percentage at both temperatures. PAb240 reacted with a low percentage of cells at 37 degrees C and no reaction was observed at 31 degrees C. PAb421 antibody stained a significantly lower percentage of cells at 37 degrees C than at 31 degrees C. Cells were not stained with PAb1620 antibody and were negative for antibodies against p21(WAF1) and MDM2 proteins independently of the temperature. Sequencing of all coding exons of the p53 gene demonstrated only a neutral genetic polymorphism, i.e. a G-to-A substitution (GAG to GAA) at nucleotide position 13 432. Thus, the observed temperature sensitivity of OvBH-1 cells cannot be ascribed to a p53 primary structure mutation. Based upon immunochemical analyses, we consider, however, that p53 in nuclei of OvBH-1 cells is in a highly unstable conformation. Furthermore, the N-terminal portion of the p53 protein at Ser20 has not been modified, and Lys373 and / or Ser378 of the C-terminus is acetylated and / or phosphorylated. The nuclear location signal of p53 is preserved. Induction of MDM2 protein is uncoupled from the cell regulatory machinery and the induction of p21(WAF1) by p53 is impaired in OvBH-1 cells.

Relations between immunologically different p53 forms, p21(WAF1) and PCNA expression in ovarian carcinomas.

The role of tumor suppressor p21WAF1 expression in epithelial ovarian cancer has not been definitely explained and the clarification of mutual p53 and p21WAF1 relations considering proliferative activity seems to be very important for understanding of a functional link between p53 and cell-cycle control. Therefore the expression of p53 and p21WAF1 was assessed immunohistochemically in a series of 50 ovarian carcinomas considering clinicopathological variables. The reactivity of three anti-p53 monoclonal antibodies (DO-7, PAb240, PAb1620) recognizing immunologically distinct forms of p53 were analysed in relation to p21WAF1 level in individual patients. p21WAF1 was expressed in 24 (48%) of all cases. The detection of p53 protein was related to the antibody applied and DO-7 antibody appears to be better than both PAb240 and PAb1620. However, independently of antibody used significant inter- and intratumoral heterogeneity in p53 and p21WAF1 expression was revealed. The identification of different p53/p21WAF1 phenotypes reflect the complex and multiple relations between these two cell-cycle regulators indicating that in ovarian carcinomas p21WAF1 activation may be both p53-dependent and p53-independent. High cell proliferation was usually accompanied by undetectable or weak p21WAF1 staining. There was no significant correlation between p53 and p21WAF1 expression and histology, stage and grade of ovarian carcinomas (p>0.05).

The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H.

Cyclin dependent kinases are regulated by phosphorylation and dephosphorylation of the catalytic cdk subunits, by assembly with specific cyclins and by specific inhibitor molecules. Recently, it turned out that cyclins are also phosphoproteins, which means that they are also potential targets for a regulation by phosphorylation and dephosphorylation. Here, we show that cyclin H was phosphorylated by protein kinase CK2. Like most other CK2 substrates cyclin H was much better phosphorylated by the CK2 holoenzyme than by the alpha-subunit alone. By using point mutants derived from the cyclin H sequence we mapped the CK2 phosphorylation site at threonine 315 at the C-terminal end of cyclin H. Phosphorylation at this position had no influence on the assembly of the cyclin H/cdk7/Mat1 complex. However, phosphorylation at amino acid 315 of cyclin H turned out to be critical for a full cyclin H/cdk7/Mat1 kinase activity when the CTD peptide of RNA polymerase II or cdk2 was used as a substrate.

Cyclin H is a new binding partner for protein kinase CK2.

The protein kinase CK2 holoenzyme is composed of two regulatory beta- and two catalytic alpha- or alpha(')-subunits. There is ample evidence for the binding of individual subunits of CK2 to various cellular proteins and, moreover, for functions of the individual subunits, which are different from their roles in the holoenzyme. Here, we report that the regulatory cyclin H subunit of the cyclin H/cdk7/Mat1 complex was associated with a protein kinase activity, which shows some similarity with protein kinase CK2. Coimmunoprecipitation experiments supported the existence of complexes of cyclin H and CK2 in mammalian cells. Far Western blot experiments revealed that cyclin H bound to the alpha-subunit but not the alpha(')- and beta-subunits of CK2. Immunofluorescence analysis showed that cyclin H and CK2alpha were colocated in the nucleus. Although cyclin H functions as the regulatory subunit for the cyclin H/cdk7/Mat1 complex, it could not substitute the regulatory beta-subunit of CK2 in its regulatory function of the CK2 activity.