Dipeptidyl peptidase IV in two human glioma cell lines.

There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III) and U87 glioblastoma multiforme (Grade IV) lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further studies of function of this enzyme in human glioma cells.     

Molecular analysis of chromosome 9q deletions in two Gorlin syndrome patients.

Gorlin syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, medulloblastomas, ovarian fibromas, and a variety of developmental defects. All affected individuals share certain key features, but there is significant phenotypic variability within and among kindreds with respect to malformations. The gene (NBCCS) maps to chromosome 9q22, and allelic loss at this location is common in tumors from Gorlin syndrome patients. Two recessive cancer-predisposition syndromes, xeroderma pigmentosum group A (XPAC) and Fanconi anemia group C (FACC), map to the NBCCS region; and unusual, dominant mutations in these genes have been proposed as the cause of Gorlin syndrome. This study presents cytogenetic and molecular characterization of germ-line deletions in one patient with a chromosome 9q22 deletion and in a second patient with a deletion of 9q22-q3l. Both have typical features of Gorlin syndrome plus additional findings, including mental retardation, conductive hearing loss, and failure to thrive. That Gorlin syndrome can be caused by null mutations (deletions) rather than by activating mutations has several implications. First, in conjunction with previous analyses of allelic loss in tumors, this study provides evidence that associated neoplasms arise with homozygous inactivation of the gene. In addition, dominant mutations of the XPAC and FACC1 genes can be ruled out as the cause of Gorlin syndrome, since the two patients described have null mutations. Finally, phenotypic features that show variable expression must be influenced by genetic background, epigenetic effects, somatic mutations, or environmental factors, since these two patients with identical alterations (deletions) of the Gorlin syndrome gene have somewhat different manifestations of Gorlin syndrome.     

Homozygous deletions on the short arm of chromosome 9 in ovarian adenocarcinoma cell lines and loss of heterozygosity in sporadic tumors.

Rat ovarian surface epithelial cells transformed spontaneously in vitro have been found to have homozygous deletions of the interferon alpha (IFNA) gene. This suggests that inactivation of a tumor-suppressor gene in this region may be crucial for the development of ovarian cancer. We therefore used microsatellite markers and Southern analysis to examine the homologous region in humans--the short arm of chromosome 9--for deletions in sporadic ovarian adenocarcinomas and ovarian tumor cell lines. Loss of heterozygosity occurred in 34 (37%) of 91 informative sporadic tumors, including some benign, low-malignant-potential and early-stage tumors, suggesting that it is an early event in the development of ovarian adenocarcinoma. Furthermore, homozygous deletions on 9p were found in 2 of 10 independent cell lines. Deletion mapping of the tumors and lines indicates that the candidate suppressor gene inactivated as a consequence lies between D9S171 and the IFNA locus, a region that is also deleted in several other tumors and that contains the melanoma predisposition gene, MLM.     

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.     

Apoptosis induced by selenium in human glioma cell lines

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NTI4 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.     

Molecular processes of chromosome 9p21 deletions causing inactivation of the p16 tumor suppressor gene in human cancer: deduction from structural analysis of breakpoints for deletions

Chromosome interstitial deletion (i.e., deletion of a chromosome segment in a chromosome arm) is a critical genetic event for the inactivation of tumor suppressor genes and activation of oncogenes leading to the carcinogenic conversion of human cells. The deletion at chromosome 9p21 removing the p16 tumor suppressor gene is a genetic alteration frequently observed in a variety of human cancers. Thus, structural analyses of breakpoints for p16 deletions in several kinds of human cancers have been performed to elucidate the molecular process of chromosome interstitial deletion consisting of formation of DNA double strand breaks (DSBs) and subsequent joining of DNA ends in human cells. The results indicated that DSBs triggering deletions in lymphoid leukemia are formed at a few defined sites by illegitimate action of the RAG protein complex, while DSBs in solid tumors are formed at unspecific sites by factors unidentified yet. In both types of tumors, the intra-nuclear architecture of chromatin was considered to affect the susceptibility of genomic segments of the p16 locus to DSBs. Broken DNA ends were joined by non-homologous end joining (NHEJ) repair in both types of tumors, however, microhomologies of DNA ends were preferentially utilized in the joining in solid tumors but not in lymphoid leukemia. The configuration of broken DNA ends as well as NHEJ activity in cells was thought to underlie the features of joining. Further structural analysis of other hot spots of chromosomal DNA breaks as well as the evaluation of the activity and specificity of NHEJ in human cells will elucidate the mechanisms of chromosome interstitial deletions in human cells.     

Origin of chromosome rearrangements in two long-lived human keratinocyte lines

Summary We have determined the origin of the extra chromosomal material in the karyotypes of two spontaneously-occurring long-lived human keratinocyte lines, HKC-N2 and HKC-N6. In each case the extra material was derived from the chromosome on which it was located. Possible relationships between the triplication of chromosomal material and the overcoming of cell senescence are discussed.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.     

Characteristics of alpha-adrenoceptors in two human colorectal cancer cell lines.

The DiFi and HT-29 human colorectal cancer cell lines were characterized and compared with respect to binding properties of alpha adrenoceptors present on the cell surface. Both cell lines possessed alpha-1 and alpha-2 adrenoceptors of high affinity; however, DiFi cells were rich in alpha-1 adrenoceptors, whereas HT-29 cells were rich in alpha-2 adrenoceptors. In each cell line, specificity of radioligand binding to alpha-1 or alpha-2 adrenoceptors was proved via competition studies using non-tritiated drugs. We believe this to be the first characterization of alpha-1 adrenoceptors in cell line HT-29 and of alpha-1 and alpha-2 adrenoceptors in DiFi cells. Differences between these cell lines in alpha adrenoceptor expression are discussed in relation to colon carcinogenesis. The high level of alpha-1 adrenoceptors seen in DiFi cells should make this cell line useful in studies of the function and regulation of this adrenoceptor subtype.     

The ultrastructure of the chromosome periphery in human cell lines

We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled interchromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.by D. Schweizer     

Specific chromosome aberrations in senescent fibroblast cell lines derived from human embryos.

In senescent fibroblast cell lines derived from human embryos, the number of chromosome aberrations were found to increase rapidly. In addition to an increase in aneuploidy and polyploidy, a high frequency of dicentrics occurred, but the number of other chromosome abnormalities remained approximately constant. Banding revealed that many of the dicentrics appeared to be end-to-end fusions of whole chromosomes. The involvement of chromosomes was nonrandom. This "telomeric binding" may reflect a progressive decrease in the stability of telomeric sequences or associated enzymes which may also occur in vivo.     

p27 modulates cell cycle progression and chemosensitivity in human malignant glioma.

The cell cycle regulatory protein p27, an inhibitor of cyclin-dependent kinases (CDK), has been attributed a role in (i) prognosis in breast and colon cancer, (ii) induction of apoptosis in cancer cells, and (iii) resistance to cancer chemotherapy. Here we report that p27 is widely expressed in human malignant gliomas in vivo and in glioma cell lines in vitro. Serum deprivation or confluency promotes p27 protein accumulation in vitro. Neither baseline p27 levels nor p27 levels induced by confluency or serum deprivation correlate with p53 status or drug sensitivity of human glioma cell lines. Expression of antisense p27 mRNA increased the doubling times in T98G glioma cells, whereas sense p27 mRNA had no such effect. There was a density-dependent and drug-specific modulation of chemosensitivity by sense or antisense mRNA expression in T98G cells. Taken together, these data define a strong p27 response to altered growth conditions and suggest a role for p27 in modulating response to chemotherapy in human malignant glioma cells.     

Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease.

Epithelial cancers inflict a heavy human and social burden. It was estimated [Tyczynski JE, Bray F, Parkin DM. Lung cancer in Europe in 2000: epidemiology, prevention, and early detection. Lancet Oncol 2003;4:45-55 (Review)] that in Europe, in the year 2000, 347 000 persons died of lung cancer alone, the deadliest cancer disease. Loss of heterozygosity and large homozygous deletions of the human chromosome region 3p21 are especially frequent in epithelial cancers of several organs. In fact, 3p21 is a very peculiar region of the genome harbouring, tightly clustered, several types of cancer-causing genes (CCG) (Lerman MI, Minna JD. The 630 kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumour suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 2000;60:6116-33). Current results show that, unlike it was thought initially, many tumour suppressor genes (TSG) are located close by even in small genomic regions. They may be involved, perhaps with varying role, in different types of tumour, and may be influenced by the genetic background of different human populations as well as by environmental pollutants (cigarette smoking, professional exposure). This review will discuss methods of molecular analysis of genomic deletions to uncover CCG at 3p21, will summarize the present knowledge regarding the TSGs located in this region, and will describe a possible model of epithelial cancer pathogenesis.     

Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.     

Prevalent involvement of illegitimate V(D)J recombination in chromosome 9p21 deletions in lymphoid leukemia

To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2B loci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, and CDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.     

Determination of eight elements in six human cancer cell lines and two human normal cell lines by PIXE

Gastric, hepatic, and pulmonary cancer cell lines, and the third passage of normal gastric and pulmonary cell lines were analyzed by proton induced X-ray emission (PIXE) method. The contents of element Sr, Ca, Fe, Zn, P, K, Cu, and As in the cell lines were determined. The Sr, Ca, Fe, Zn, and As contents in cancer cell lines were significantly lower than those in the normal cell lines (p less than 0.05), whereas there were no significant differences for the P, K, and Cu contents (p greater than 0.1). The results suggest that the need of some essential elements has been diminished in cancer cell proliferation.