Markus Seifert

Glyceraldehyde-3-phosphate Dehydrogenase and Nm23-H1/Nucleoside Diphosphate Kinase A TWO OLD ENZYMES COMBINE FOR THE NOVEL Nm23 PROTEIN PHOSPHOTRANSFERASE FUNCTION

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH·Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteinsin vitro. The molecular mass of the highly purified recombinant GAPDH·Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

Expression of calpain I messenger rna in human renal cell carcinoma : Correlation with lymph node metastasis and histological type

Calpain, also named CANP (for calcium‐activated neutral protease), is an intracellular cytoplasmatic non‐lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c‐Fos and c‐Jun, the tumor supressor protein p53, protein kinase C, pp60c‐src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL 1) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern‐blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression. Int. J. Cancer (Pred. Oncol.) 84:6–9, 1999.

Vitamin D3 Metabolism in Human Glioblastoma Multiforme: Functionality of CYP27B1 Splice Variants, Metabolism of Calcidiol, and Effect of Calcitriol

Purpose: A better understanding of the vitamin D3 metabolism is required to evaluate its potential therapeutic value for cancers. Here, we set out to contribute to the understanding of vitamin D3 metabolism in glioblastoma multiforme. Experimental Design: We did nested touchdown reverse transcription-PCR (RT-PCR) to identify CYP27B1 splice variants and real-time RT-PCR to quantify the expression of CYP27B1. A cell line was treated with calcitriol to determine the effect on the expression of CYP27B1, 1α,25-dihydroxyvitamin D3-24-hydroxylase (CYP24), and vitamin D3 receptor (VDR). We generated three antibodies for the specific detection of CYP27B1 and splice variants. High-performance TLC was done to determine the endogenous CYP27B1 activity and the functionality of CYP27B1 splice variants. Using WST-1 assay, we determined the effect of vitamin D3 metabolites on proliferation. Results: We report a total of 16 splice variants of CYP27B1 in glioblastoma multiforme and a different expression of CYP27B1 and variants between glioblastoma multiforme and normal tissues. We found preliminary evidence for enzymatic activity of endogenous CYP27B1 in glioblastoma multiforme cell cultures but not for the functionality of the splice variants. By adding calcitriol, we found a proliferative effect for some cell lines depending on the dose of calcitriol. The administration of calcitriol led to an elevated expression of CYP27B1 and CYP24 but left the expression of the VDR unaltered. Conclusions: Our findings show that glioblastoma multiforme cell lines metabolize calcidiol. In addition, we show various effects mediated by calcitriol. We found a special vitamin D3 metabolism and mode of action in glioblastoma multiforme that has to be taken into account in future vitamin D3–related therapies.

In vitro comparison of the vitamin D endocrine system in 1,25(OH)2D3-responsive and -resistant melanoma cells.

We studied effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], its analog seocalcitol (EB 1089), and 25-hydroxyvitamin D3 [25(OH)D3], on the growth of seven melanoma cell lines. While three cell lines (MeWo, SK-Mel-28, SM) responded to antiproliferative effects of active vitamin D analogs, the others (SK-Mel-5, SK-Mel-25, IGR, MelJuso) were resistant. A strong induction (7000-fold) of 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase, CYP24) mRNA was detected in responsive cell lines along with 1,25(OH)2D3-treatment, indicating functional integrity of vitamin D receptor (VDR)-mediated transcription. In contrast, induction of 24OHase was much lower in resistant melanoma cells (70-fold). VDR mRNA was induced up to 3–fold both in responsive and resistant cell lines along with 1,25(OH)2D3-treatment. RNA for vitamin D-activating enzymes vitamin D-25-hydroxylase (25OHase, CYP27A1) and 25-hydroxyvitamin D-1α-hydroxylase (1αOHase, CYP27B1) was detected in all melanoma cell lines analyzed, additionally we show splicing variants of 1αOHase in SK-Mel-28 cells. Expression of 25OHase and 1αOHase was marginally modulated along with treatment. Proliferation of melanoma cells was not inhibited by treatment with 25(OH)D3, indicating no significant stimulation of endogeneous production of antiproliferative acting 1,25(OH)2D3. In conclusion, we characterize the vitamin D endocrine system in melanoma cells and demonstrate that the majority of melanoma cell lines analyzed is resistant to antiproliferative effects of 1,25(OH)2D3. It can be speculated that these alterations are of importance for pathogenesis and growth of metastasizing malignant melanoma. Additionally, our findings indicate that only a minority of cases with metastasizing melanoma may represent a promising target for palliative treatment with new vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of 1,25(OH)2D3-synthesis/metabolism.

Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin

Calpain, also named CAPN (for calcium‐activated neutral protease), is a ubiquitous intracellular cytoplasmic non‐lysosomal cysteine endopeptidase that requires calcium ions to exert its activity. Two major isoenzymes are known—µ‐calpain (CAPN1) and m‐calpain (CAPN2)—requiring micromolar and millimolar calcium concentrations for activation, respectively. Many known substrates of the different calpain isoenzymes, such as the transcription factors c‐Fos and c‐Jun, the tumour suppressor protein p53, protein kinase C, pp60src, or the adhesion molecule integrin, have been implicated in the pathogenesis of various malignancies including squamous (SCC) and basal (BCC) cell carcinomas of human skin, suggesting an important role of the calpain isoenzymes in malignant diseases. We have analysed the expression of CAP1 and CAPN2 protein and mRNA expression in BCCs and SCCs of human skin. Interestingly, CAPN1 immunoreactivity (streptavidin–peroxidase technique) was markedly reduced in BCCs compared to normal human skin or SCCs, while in contrast CAPN1 mRNA levels (determined by real‐time PCR) were markedly elevated in BCCs and SCCs compared to normal human skin. No differences were found analysing CAPN2 protein and mRNA expression in normal human skin, BCCs and SCCs. In conclusion, we have demonstrated for the first time alterations in calpain mRNA expression and protein content in malignant skin tumours that may be of importance for the tumorigenesis and growth characteristics of BCCs and SCCs. However, our results do not allow conclusions on the function of CAPN1 and CAPN2 in BCCs and SCCs. It is not known if the CAPN genes in BCCs or SCCs exhibit functionally inactivating mutations or whether decreased CAPN1 protein expression in BCCs and elevated CAPN1 mRNA in BCCs and SCCs reflect a feedback loop coupled with increased degradation or proteolysis of CAPN1 protein. Copyright

1[alpha],25‐dihydroxyvitamin D3 enhances annexin II dependent proliferation of osteoblasts

Cells experience a variety of physiological and non‐physiological stresses and consequently have appropriate mechanisms to deal with such deviations from homeostasis. Particularly subject to mechanical stress and shear forces are the cells that make up the bones. Osteoblastic cells can interpret this stress as a stimulus for proliferation; however, the molecular mechanisms underlying this phenomenon are poorly understood. We have identified annexin II as being specifically upregulated in mechanically stressed osteoblasts and found that increased levels of this protein are necessary for 1[alpha],25‐dihydroxyvitamin D3 mediated augmentation of the proliferative response of osteoblasts after mechanical stress. Our data demonstrate a novel interaction between 1[alpha],25‐dihydroxyvitamin D3 and annexin II in the proliferative response of osteoblasts as well as a novel function for annexin II in the stress response. These findings may offer new therapeutic opportunities for conditions that require regenerative osteoblastic activity such as osteoporosis. J. Cell. Biochem. 100: 679–692, 2007.

Reciprocal responses of fibroblasts and melanocytes to α-MSH depending on MC1R polymorphisms.

The melanocortin 1-receptor (MC1R) exhibits several variants in form of single nucleotide polymorphisms (SNPs) which are known to differentially regulate melanocyte function. However, whether and how MC1R polymorphisms also affect fibroblast function, has not been investigated so far. Therefore we measured intracellular cyclic adenosine monophosphate (cAMP) concentrations (cAMP-EIA) and cellular proliferation (CellTiter-Blue) upon stimulation with alpha-melanocyte stimulating hormone (α-MSH) in eight different human fibroblast and melanocyte cell lines with wild type and different MC1R SNPs. We found that fibroblasts, as well as melanocytes, show differences in MC1R function depending on the MC1R genotype. MC1R stimulation with α-MSH in wild type (MC1Rwt) melanocytes results in an increase of intracellular cAMP and cellular proliferation. In contrast, MC1Rwt fibroblasts react with a decrease of intracellular cAMP and proliferation. In MC1R polymorphic fibroblasts (R163Q, R151C and V60L) both effects are significantly alleviated. Similar, but inverse effects could be found in MC1R polymorphic melanocytes (R142H and V92M) with a significantly lower cAMP increase and proliferation rate compared to MC1Rwt melanocytes. Our results indicate that the MC1R displays reciprocal growth responses in melanocytes and fibroblasts, depending on the MC1R genotype. Thus, the MC1R seems to be not solely important for the skin pigmentary system, but also for the fibroblast function, and might influence different processes of the dermal compartment like wound healing, fibrosis and keloid formation.

Modulation of Vitamin D-induced growth inhibition in melanoma cell lines: implications for an important function of vitamin D receptor (VDR) and 1,25-dihydroxyvitamin D3-24-hydroxylase (24-OHase) expression, histonedeacetylation, and calpain activity

Increasing evidence indicates an important role of the skin vitamin D system for tumorigenesis and progression of malignant melanoma. We have now characterized the vitamin D system in melanoma cell lines, detecting strong RNA expression of vitamin D receptor (VDR), 25‐hydroxyvitamin D‐1α‐hydroxylase (1αOHase), vitamin D‐25‐hydroxylase (25OHase) and 1,25‐dihydroxyvitamin D‐24‐hydroxylase (24OHase) in various melanoma cell lines (MEWO, SKMEL28, BU47HOM) using real time PCR (LightCycler) and specific hybridisation probes. We then have analyzed effects of 1,25‐dihydroxyvitamin D3 and analogs (EB 1089, MC 1288) on proliferation and apoptosis as well as on expression of VDR, 1αOHase), 25OHase, and 24OHase in various melanoma cell lines (BUHOM, MEWO, SKMEL)in vitro. Using a tetrazolium salt (WST‐1) based colorimetric assay, we detected dose‐dependent inhibition of cell growth (up to 40%) induced by calcitriol or its analogs. Treatment of melanoma cells with calcitriol resulted in decreased immunoreactivity of proliferation markers including Ki‐67 and PCNA. Flow cytometry experiments (bcl‐2, bcl‐xl, bcl‐xs, bcl‐x, bax, CD95) and analysis of annexin Pi expression revealed no induction of apoptosis by calcitriol (10−7 M) or its analogs, while increased levels of bcl‐2 protein were detected. Additionally, we show modulation of vitamin D‐induced inhibition of cell proliferation by calpain inhibitors I and II (Calbiochem) as well as by inhibitors of histonedeacetylation (sodium butyrate, trichostatin A). In conclusion our findings indicate that (i) vitamin D analogs suppress proliferation but do not induce apoptosis in melanoma cell lines, (ii) induction of VDR and 24‐OHase expression as well as histonedeacetylation and calpain activity modulate vitamin D‐induced growth inhibition, (iii) new calcitriol analogs exerting less systemic side effects may be interesting candidates for the treatment of metastasizing malignant melanoma.