Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein

Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.     

Novel reference gene RPN1 for normalization of quantitative data in lung and kidney cancer

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different mRNA/cDNA samples with unknown concentration. For this purpose reference genes with stable expression level (such as GAPDH, ACTB, HPRT1, TBP) are used. The choice of appropriate reference genes is still actual because well-known reference genes are not suitable for certain cancer types frequently and their unreasonable use without additional tests lead to wrong conclusions. We have developed the bioinformatic approach and selected a new potential reference gene RPN1 for lung and kidney tumors. This gene is located at the long arm of chromosome 3. Our method includes mining of the dbEST and Oncomine databases and functional analysis of genes. The RPN1 was selected from 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI-microarrays we found no methylation, deletions and/or amplifications at the RPN1-containing locus in 56 non-small cell lung and 42 clear cell renal cancer samples. Using RT-qPCR we showed low variability of RPN1 mRNA level comparable to those of reference genes GAPDH and GUSB in lung and kidney cancer. The mRNA levels of two target genes coding hyalouronidases--HYAL1 and HYAL2--were estimated and normalized relative to pair RPN1--GAPDH genes for lung cancer and RPN1--GUSB for kidney cancer. These combinations were shown to be optimal for obtaining accurate and reproducible data. All obtained results allow us to suggest RPN1 as novel reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.     

Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level

In this work for the first time copy number and expression changes of the tumor suppressor gene RBSP3 (3p21.3) were investigated. The study was performed on HPV-positive squamous cervical carcinoma (SCC) using real-time PCR. Deletions were detected in 42% of cases (19 of 45 studied biopsies). Frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P < 0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). Expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P < 0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparison of copy number and expression changes showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes in copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC and complex mechanisms for inactivation of RBSP3. We also hypothesize that these data indicate that RBSP3 in addition to dephosphorylation of pRb has other functions important for malignant transformation because pRb is almost absent in HPV-positive SCC.     

Isolation and some properties of homogenous nuclease S1 from alpha-amyloryzin

A purification method for isolating homogeneous single-strand specific nuclease S1 from alpha-amyloryzin has been developed. The yield was about 16% and purification factor--9000. Nuclease S1 thus obtained was proved to be free of contaminations of any others nucleolytic enzymes. It is shown for the first time that ribo- and deoxy-dinucleosidemonophosphates are hydrolyzed by nuclease S1 to form 5'-nucleotides with pH optimum for ApA equal to 4.6.     

<Emphasis Type="Italic">RPN1</Emphasis>, a new reference gene for quantitative data normalization in lung and kidney cancer

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different specimens with unknown mRNA/cDNA concentrations. For this purpose, reference genes with stable expression are used (e.g., GAPDH, ACTB, HPRT1, or TBP). The problem of choosing proper reference genes is still a topical issue, because well-known reference genes can be unsuitable for certain cancer types and their inappropriate use without additional testing can lead to wrong conclusions. A recently developed bioinformatical approach was employed to identify a new potential reference gene for lung and kidney tumors, RPN1, located on the long arm of chromosome 3. The method employed the mining of the dbEST and Oncomine databases and functional analysis of genes. RPN1 was selected from approximately 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI microarrays, we found no methylation, deletions, and/or amplifications in the RPN1-containing locus in 56 nonsmall cell lung and 42 clear cell renal cell cancer specimens. Real-time PCR showed that variation of RPN1 mRNA levels in nonsmall cell lung cancer and clear-cell renal cancer was low and comparable to that of the known reference genes GAPDH and GUSB, respectively. Expression levels of two hyalouronidase genes, HYAL1 and HYAL2, were assessed using the suggested references gene pairs (RPN1-GAPDH for lung cancer and RPN1-GUSB for kidney cancer), and these combinations were shown to produce accurate and reproducible data. These results suggest that RPN1 is a new, promising reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.