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PubMed1992

Cellular expression of tropoelastin mRNA splice variants.

The primary transcript of tropoelastin is alternatively spliced into multiple mRNAs. The pattern and frequency of exon splicing is developmentally regulated, but the cellular profile of isoform expression within and among elastic tissues is not known. We used splice-variant specific antisense oligomeric deoxyribonucleotide probes in an in situ hybridization assay to assess the distribution of cells undergoing specific alternative splicing of tropoelastin pre-mRNA in developing bovine elastic tissues. Antisense oligomers were synthesized to exon sequences that are not alternatively spliced (exon 36) and to sequences that become abutted after high frequency (exon 33) and low frequency (exons 13 and 14) alternative splicing. The specificity of these probes for tropoelastin splice variants was verified by Southern hybridization to tropoelastin cDNAs with known exon deletions, and their specificity for tropoelastin mRNA was demonstrated by Northern hybridization. In situ hybridization with [35S]-labeled oligomers on sections of bovine lobar pulmonary artery and other elastic tissues showed that all elastogenic cells produce multiple forms of tropoelastin mRNA. These observations suggest that the production of tropoelastin isoforms is common to all cells within an elastin tissue and that this multiplicity may not be involved in regional differences in elastic tissue architecture.

Animals↗

PubMed2006

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload.

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

Calcium↗

PubMed1997

[Diagnostic value of CD44 splice variants in urine exfoliated cells of bladder cancer].

To study the early and non-invasive diagnostic value of CD44 splice variants in urine exfoliated cells of bladder cancer. We used reverse transcriptionpolymerase chain resection (RT-PCR) and southern blot hybridization to detect CD44 splice variants in exfoliated cells in 40 urine samples (20 bladder cancers, 20 non-neoplastic controls), and compared with the results of urine cytology on the same set of samples. 90% (18/20) of the urine samples of bladder cancer showed overexpression of CD44 splice variants while none of the 20 controls did so. This method not only has a sensitivity of 90% (18/20) which was much higher than that of 65% (13/20) by using urine cytology, but also is non-invasive and comfortable. The results suggest that CD44 splice variants in exfoliated cells in urine samples are a new tumor marker for early and non-invasive diagnosis of bladder cancer.

Biomarkers, Tumor↗

PubMed2006

Specific use of start codons and cellular localization of splice variants of human phosphodiesterase 9A gene.

BACKGROUND: Phosphodiesterases are an important protein family that catalyse the hydrolysis of cyclic nucleotide monophosphates (cAMP and cGMP), second intracellular messengers responsible for transducing a variety of extra-cellular signals. A number of different splice variants have been observed for the human phosphodiesterase 9A gene, a cGMP-specific high-affinity PDE. These mRNAs differ in the use of specific combinations of exons located at the 5' end of the gene while the 3' half, that codes for the catalytic domain of the protein, always has the same combination of exons. It was observed that to deduce the protein sequence with the catalytic domain from all the variants, at least two ATG start codons have to be used. Alternatively some variants code for shorter non-functional polypeptides. RESULTS: In the present study, we expressed different splice variants of PDE9A in HeLa and Cos-1 cells with EGFP fluorescent protein in phase with the catalytic domain sequence in order to test the different start codon usage in each splice variant. It was found that at least two ATG start codons may be used and that the open reading frame that includes the catalytic domain may be translated. In addition the proteins produced from some of the splice variants are targeted to membrane ruffles and cellular vesicles while other variants appear to be cytoplasmic. A hypothesis about the functional meaning of these results is discussed. CONCLUSION: Our data suggest the utilization of two different start codons to produce a variety of different PDE9A proteins, allowing specific subcellular location of PDE9A splice variants.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5) and altered Axin-1 expression during tumour progression.

BACKGROUND: Splicing processes might play a major role in carcinogenesis and tumour progression. The Wnt pathway is of crucial relevance for cancer progression. Therefore we focussed on the Wnt/beta-catenin signalling pathway in order to validate the expression of sequences predicted as alternatively spliced by bioinformatic methods. Splice variants of its key molecules were selected, which may be critical components for the understanding of colorectal tumour progression and may have the potential to act as biological markers. For some of the Wnt pathway genes the existence of splice variants was either proposed (e.g. beta-Catenin and CTNNB1) or described only in non-colon tissues (e.g. GSK3beta) or hitherto not published (e.g. LRP5). RESULTS: Both splice variants--normal and alternative form--of all selected Wnt pathway components were found to be expressed in cell lines as well as in samples derived from tumour, normal and healthy tissues. All splice positions corresponded totally with the bioinformatical prediction as shown by sequencing. Two hitherto not described alternative splice forms (CTNNB1 and LRP5) were detected. Although the underlying EST data used for the bioinformatic analysis suggested a tumour-specific expression neither a qualitative nor a significant quantitative difference between the expression in tumour and healthy tissues was detected. Axin-1 expression was reduced in later stages and in samples from carcinomas forming distant metastases. CONCLUSION: We were first to describe that splice forms of crucial genes of the Wnt-pathway are expressed in human colorectal tissue. Newly described splicefoms were found for beta-Catenin, LRP5, GSK3beta, Axin-1 and CtBP1. However, the predicted cancer specificity suggested by the origin of the underlying ESTs was neither qualitatively nor significant quantitatively confirmed. That let us to conclude that EST sequence data can give adequate hints for the existence of alternative splicing in tumour tissues. That no difference in the expression of these splice forms between cancerous tissues and normal mucosa was found, may indicate that the existence of different splice forms is of less significance for cancer formation as suggested by the available EST data. The currently available EST source is still insufficient to clearly deduce colon cancer specificity. More EST data from colon (tumour and healthy) is required to make reliable predictions.

Aged↗

PubMedDate not supplied

SHP-1 variant proteins are absent in motheaten mice despite presence of splice variant transcripts with open reading frames.

Motheaten mice have a mutation that causes abnormal splicing of the SHP-1 gene producing transcripts that are out of frame. Thus, motheaten mice cannot produce normal SHP-1 protein. However, we have found that the SHP-1 locus in normal mice is expressed as multiple in-frame splice variant transcripts. We report here that the motheaten SHP-1 gene is likewise expressed in multiple spliced forms, two of which are in frame. One of these two variants, which is also present in normal mice, lacks the exon containing the motheaten mutation and is therefore expected to encode an active phosphatase with only one of the two SH2 domains of SHP-1. We showed that both of these variants produce phosphatases with a higher specific activity than SHP-1, suggesting that motheatein mice are not SHP-1 null. The possibility that motheaten mice produce disregulated phosphatases offered a simple explanation for the puzzling observation that substrates of SHP-1 are hypo-phosphorylated in motheaten mice. We tested this by measuring for SHP-1 protein and activity in motheaten macrophages. However, we did not detect specific activity, and found that one of these variant proteins was unstable. These findings likewise suggest that little or no SHP-1 variant proteins exist in normal cells.

Alternative Splicing↗

PubMed2001

Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease protection assays.

The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.

Alternative Splicing↗

PubMed1999

Effects of chronic alcohol consumption on the expression of different NR1 splice variants in the brain of AA and ANA lines of rats.

Following chronic alcohol treatment alterations in N-methyl-D-aspartate receptor subunit 1 and 2 (NR1 and NR2), mRNA and protein levels have been reported. The NR1 gene undergoes alternative RNA splicing, resulting in eight splice variants, which were shown to differ in their sensitivity to alcohol. Here, we studied mRNA and protein levels of NR1 splice variants in alcohol-preferring (AA) and alcohol-nonpreferring (ANA) rat lines under basal conditions (alcohol-naive), and following chronic alcohol consumption. mRNA levels of three NR1 splice variants (NR1-1, NR1-2, NR1-4), and the protein levels of NR1 (NR1-1/NR1-2), and of NR1 alternative C-terminus (NR1-3/NR1-4) were determined in the hippocampus and nucleus accumbens by competitive RT-PCR and Western blot analysis, respectively. No significant differences in NR1 mRNA, or protein levels were found in the nucleus accumbens between the two rat lines under basal conditions, or following chronic alcohol consumption. In the hippocampus of alcohol-naive rats, the NR1-4 mRNA content was significantly higher in ANA compared to AA rats, however, no significant difference could be detected at the protein level. Following chronic alcohol consumption, the protein level of the NR1 alternative C-terminus (NR1-3/NR1-4) was significantly higher in AA rats compared to the corresponding control. Taken together, these results suggest: (i) brain site-specific alterations in NMDA receptor subunit composition occur following chronic alcohol consumption. (ii) In the hippocampus, NR1 splice variant mRNA levels differ between AA and ANA rats. (iii) The mRNA levels and protein levels of NR1 splice variants are differentially affected by chronic alcohol consumption.

Alcohol Drinking↗

PubMed1999

The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits.

Here we report an interaction between AMPA receptor subunits and a single PDZ domain-containing protein called PICK1 which is known to bind protein kinase C alpha (PKC alpha). The interaction occurs within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif. The PDZ domain of PICK1 is required for the interaction and the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1 and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha indicating that the same domain of PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2; short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals colocalization and an altered pattern of distribution for each protein from when they are expressed individually. This novel interaction provides a possible regulatory mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha.

Animals↗

PubMed2002

Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men.

OBJECTIVE: To investigate whether or not alternatively spliced variants of the FSH receptor gene occur in human testis and whether the presence of the splicing variants is associated with spermatogenic defects and serum FSH concentration in infertile men. DESIGN: A prospective case control study. SETTING: An IVF clinic and infertility laboratory at a university hospital. PATIENT(S): Forty-three infertile patients undergoing testicular biopsy. INTERVENTION(S): Total RNA was extracted from the testicular tissues and used for reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S): Expression pattern was analyzed by nested RT-PCR using primers designed to amplify a fragment of FSH receptor gene. PCR products of splicing variants were cloned and sequenced. RESULT(S): The PCR products showed three kinds of additional bands corresponding to alternatively spliced isoforms of the FSH receptor gene. Exon 9 deleted variant was detected in all patients and inclusion variant of small extra exon was detected in 64% (9/14) of the patients with normal spermatogenesis and 34% (10/29) of the patients with spermatogenic defects. The presence of inclusion variant was not significantly associated with spermatogenic defects but was associated with a low level of serum FSH. On the other hand, exon 6 deleted variant was detected in only one patient having a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. CONCLUSION(S): We identified three different types of alternatively spliced variants of the human FSH receptor. However, it is not clear whether or not there is an association between three variants and spermatogenic defects.

Alternative Splicing↗

PubMed2004

Towards a complete picture of splice variants of the gene for 25-hydroxyvitamin D31alpha-hydroxylase in brain and skin cancer.

Recently, we reported amplification of the gene encoding the P450 Cytochrome 25-hydroxyvitamin D(3)1alpha-hydroxylase (CYP27B1) in 25% of human malignant glioma. Additionally, we reported the first alternative splice variants of CYP27B1. Here, we developed and employed a highly specific approach that combined nested and touchdown PCR to clone full length CYP27B1. In addition, we identified several new splice variants in human melanoma, glioblastoma multiforme (GBM), cervix carcinoma and kidney cell lines. All of the examined cell lines showed a similar expression pattern of the CYP27B1 variants. The new splice variants that were termed Hyd-V5, -V6, -V7, and -V8 were cloned and sequenced. All but one of the new variants showed an insertion of intron 1 leading to a premature termination signal and to truncated proteins without ferredoxin and haem-binding site of the P450 protein. There was no influence of 1alpha,25(OH)(2)D(3) on the expression pattern of the splice variants in melanoma cell line SkMel28.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1997

Expression of a novel splice variant of human mGluR1 in the cerebellum.

We have isolated clones of a novel splice variant of metabotropic glutamate receptor type 1 (mGluR1) from a human cerebellum cDNA library. Translation of this variant, mGluR1g would result in the addition of just one amino acid after the exon/intron boundary where the other splice variants diverge. RNA dot blot analysis using an mGluR1g-specific probe demonstrated expression in the cerebellum and also high levels in the kidney. Northern blotting using the same probe showed expression of a 4 kb transcript in the cerebellum. In situ hybridization studies in the cerebellum showed that mGluR1g mRNA is only expressed in granule cells, compared with mGluR1a/b mRNA which is also found in Purkinje cells and basket cells. Transcripts of the analogous splice variant are also present in rat brain mRNA.

Alternative Splicing↗

PubMed1999

Molecular cloning and characterization of splice variants of human RAD50 gene.

In this report, splice variants of human RAD50 (hRAD50) were cloned and characterized. A Northern blot survey identified two transcripts that hybridized to a hRAD50 cDNA clone, an upper faint band (5.9kb) and lower dense band (4.6kb). cDNA clones (hRAD50-2, 4.6kb) encompassing the entire hRAD50 transcript but having a shorter 3'-untranslated region (3'UTR) than the previously reported hRAD50-1 cDNA (5.9kb; Dolganov, G.M., Maser, R.S., Novikov, A., Tosto, L., Chong, S., Bressan, D.A., Petrini, J.H.J., 1996. Human Rad50 is physically associated with human Mre11: Identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol. Cell. Biol. 16, 4832-4841.) were isolated. The presence of AU-rich sequences in the 3'UTR of hRAD50-1, which define mRNA instability and Northern results, suggest that hRAD50-2 is the major transcript of hRAD50. A third alternative splice variant that lacks the ATP-binding domain was also identified (hRAD50-3, approximately 4.5kb). Expression of hRAD50-3 transcript was detected in all tissues examined by RT-PCR (reverse transcriptase-polymerase chain reaction) and nested DNA-PCR analyses. Expression of hRAD50 partially rescued the MMS (methyl methanesulfonate)-sensitive phenotype in rad50 mutant yeast, whereas hRAD50-3 did not show complementation. These data suggest that the hRAD50-3 does not repair DNA double-strand breaks most likely due to its inability to bind ATP, and to bind damaged DNA. The existence of these alternative splice forms is potentially important in regulation of the biological activity of the DNA recombinational repair gene, hRAD50.

3' Untranslated Regions↗

PubMed2005

Insulin-like growth factor-1 gene splice variants as markers of muscle damage in levator ani muscle after the first vaginal delivery.

OBJECTIVE: Studies in animals and human muscle have demonstrated differential splicing of the insulin-like growth factor-1 gene in response to mechanical strain and damage. We conducted a study on the expression of insulin-like growth factor-1 splice variants in the levator ani muscle after the first vaginal delivery. STUDY DESIGN: Ten women were recruited after the first vaginal delivery. Biopsy specimens were taken vaginally of the pubovisceral component of the levator ani muscle. Five nonpregnant women were recruited as control subjects. Samples were processed with real-time quantitative polymerase chain reaction, with specific primers for the insulin-like growth factor-1 splice variants. RESULTS: Insulin-like growth factor splice variants mechano growth factor and insulin-like growth factor-1Ea were significantly up-regulated (100- and 1000-fold) in the delivery population, compared with control subjects (P=.012 and .04, respectively). Statistical analysis indicated a correlation between the expression of the insulin-like growth factor-1 splice variants and the length of the second stage. CONCLUSION: These results show that damaged levator ani muscle results from stretch and overload after the first vaginal delivery.

Adult↗

PubMed1999

Splice variants of the nuclear dot-associated Sp100 protein contain homologies to HMG-1 and a human nuclear phosphoprotein-box motif.

Sp100 and PML are interferon-inducible proteins associated with a new class of nuclear domains (known as nuclear dots or PML bodies) which play a role in tumorigenesis, virus infections, and autoimmunity. While PML is extensively alternatively spliced, only two splice variants are known for Sp100. Here we describe the identification and characterization of several Sp100 splice variant proteins and support their existence by elucidation of the 3'-end of the Sp100 gene. Some of the splice variants contain a domain of significant sequence similarity with two previously described highly related interferon-inducible nuclear phosphoproteins as well as to suppressin and DEAF-1, which altogether define a novel protein motif, termed HNPP-box. One class of splice variants contains an almost complete and highly conserved copy of the DNA-binding high mobility group 1 protein sequence and thus represent novel HMG-box proteins. When expressed transiently, both major classes of Sp100 splice variant proteins localize in part to nuclear dots/PML bodies and in addition to different nuclear domains. Furthermore, PML was occasionally redistributed. These data indicate that alternatively spliced Sp100 proteins are expressed, differ in part in localization from Sp100, and might bind to chromatin via the HMG domain.

Alternative Splicing↗

PubMed2006

Analysis of the mRNA expression of the TGF-Beta family in testicular cells and localization of the splice variant TGF-beta2B in testis.

The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2, and TGF-beta3, and their receptors [TbetaRI, TbetaRII, TbetaRIII (betaglycan)] elicit many functions in the testis, for example, they perturb the blood testis barrier (BTB). Although expression of the ligands and receptors have been investigated, the alternative splice variants are incompletely examined. We therefore have analyzed all ligands, the receptors, and the splice variants TbetaRIB, TbetaRIIB, and TGF-beta2B in testicular cells from rat and mouse. In mouse, the novel transcript variant TGF-beta2B was identified and was found in Leydig cells, spermatogonia, pachytene spermatocytes, and in the apical regions of the Sertoli cells in adult testis. Even though expression of the splice variant TbetaRIB could be shown in mouse and rat, we never found the isoform TbetaRIIB in the rat cell lines studied. Whereas in all testicular cells expression of all TGF-beta ligands could be shown, receptor mRNA expression was slightly more diverse. Furthermore, expression pattern of the splice variants was more heterogeneous, for example, TbetaRIB was not detectable in adult Sertoli cells, primary peritubular cells, and immortalized peritubular cells. The heterogeneous expression of the receptors and especially of the splice variants might provide possible clues for the different functions of the TGF-beta ligands in testicular cells.

Alternative Splicing↗

PubMed2000

Differential mRNA expression of the two mineralocorticoid receptor splice variants within the human brain: structure analysis of their different DNA binding domains.

In human brain tissue, cortisol action, at basal concentrations, is mediated by the mineralocorticoid receptor (MR). An in-frame insertion of 12 bp in the MR-DNA-binding domain due to alternative splice site usage between exons 3 and 4 results in an MR mRNA splice variant (MR+4) encoding a receptor protein with four additional amino acids compared to the wild-type MR protein. To elucidate the questions of sex, age, and/or tissue dependent differences of the relative amount of the two mRNA subtypes, we examined 131 fresh human brain tissue samples from temporal and frontal lobe or hippocampus. One hundred and twenty samples were obtained from patients with epilepsy and 11 samples from patients with brain tumours. A small but significant difference of the MR+4 mRNA splice variant proportions in cortex (9.5 +/- 0.8%) and subcortical white matter (6.6 +/- 0.7%) of the temporal lobe could be detected, indicating differential MR splice variant expression within these brain areas. Moreover, the splice variant ratios in samples of the temporal lobe cortex collected from patients with epilepsy differed from samples of patients with brain tumours. These data point to an altered expression of the MR splice variants in epilepsy, and strengthen the supposition of a tissue specific alternative splicing of the MR mRNA. The frequent occurrence of the MR+4 transcript raises the question of its functional significance. For this reason, an MR+4 DNA-binding-domain structure model was generated by computer-based homology modelling based on the known glucocorticoid receptor structure. The data obtained revealed no distorting effect of the inserted four amino acids on the adjacent secondary structures, thereby suggesting that both zinc fingers retain their function. The resulting structure of the MR+4 model leads to the supposition that the receptor retains its function. Moreover, databank analysis with respect to this kind of steroid receptor variation and our own sequence data of the closely related progesterone receptor sustained the hypothesis that only corticosteroid receptors were affected by this alternative splicing event.

Adult↗

PubMedDate not supplied

Synthesis and expression of mRNA encoding for different versican splice variants is related to the aggregation of human epithelial mesothelioma cells.

Malignant mesothelioma often has a biphasic growth pattern of epithelial and/or sarcomatous morphology. In culture, epithelial cells form aggregates, whereas fibroblast-like cells do not. Two human mesothelioma cell sub-lines, one with epithelial differentiation and the other with fibroblast-like phenotype were studied. We have previously shown (Dobra et al, 2000) that distinct types of the cell-associated syndecans are involved in the regulation of mesothelioma cell differentiation, whereas the role of matrix proteoglycans (PGs) remains unknown. This study was undertaken to examine whether cell aggregation of the epithelial mesothelioma cells correlates to the differential expression of the matrix PGs versican and perlecan at different degrees of confluence. PGs were isolated from the culture medium using ion-exchange chromatography and identified by high-performance liquid chromatography, capillary electrophoresis and Western blotting. Fibroblast-like cells express substantially more versican than epithelial cells. RT-PCR showed that both cell lines express mRNA coding for versican splice variants V0 and V1, but not for V2. The dominating splice variant in both cell lines is the V0. Screening of versican splice variants in various degrees of culture confluence showed that the expression of mRNA conding for the versican splice variants V0 and V1 is different only in confluent cultures. No significant differences in the expression of perlecan between the two cell lines were recorded. These results suggest that the aggregation of epithelial cells is related to a significant decrease (p < or = 0.001) of the splice variant V1. This variant seems to be a biologically active constituent that affects tumor biology.

Alternative Splicing↗