Novel cell lines established from pediatric brain tumors

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat “fingerprints” matching that of the patient’s tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.     

Novel mdm2 splice variants identified in pediatric rhabdomyosarcoma tumors and cell lines

Mdm2 is an oncogene that binds to and inactivates the tumor suppressor p53. However, the presence of oncogenic splice variants of mdm2 in human tumors that lack the p53 binding site has suggested a p53-independent transforming function for this protein. This report describes expression of 11 different mdm2 splice variants in pediatric rhabdomyosarcoma (RMS) cell lines and tumors at a frequency of 75% and 82%, respectively. Five of these isoforms have previously been described in other tumor histiotypes but six are novel and may be unique to RMS. There was no association between expression of splice variants and mdm2 gene amplification or p53 status. In addition, the frequency of splice variants was much higher than the incidence of mdm2 amplification or p53 mutations. These variants may be important to consider with respect to RMS tumor progression and therapeutic response.     

DNA polymerase beta expression differences in selected human tumors and cell lines.

A long-standing question in cancer biology has been the extent to which DNA repair may be altered during the process of carcinogenesis. We have shown recently that DNA polymerase beta (beta-pol) provides a rate-determining function during in vitro repair of abasic sites by one of the mammalian DNA base excision repair pathways. Therefore, altered expression of beta-pol during carcinogenesis could alter base excision repair and, consequently, be critical to the integrity of the mammalian genome. We examined the expression of beta-pol in several cell lines and human adenocarcinomas using a quantitative immunoblotting method. In cell lines from normal breast or colon, the level of beta-pol was approximately 1 ng/mg cell extract, whereas in all of the breast and colon adenocarcinoma cell lines tested, a higher level of beta-pol was observed. In tissue samples, colon adenocarcinomas had a higher level of beta-pol than adjacent normal mucosa. Breast adenocarcinomas exhibited a wide range of beta-pol expression: one tumor had a much higher level of beta-pol (286 ng/mg cell extract) than adjacent normal breast tissue, whereas another tumor had the same level of beta-pol as adjacent normal tissue. Differences in beta-pol expression level, from normal to elevated, were also observed with prostate adenocarcinomas. All kidney adenocarcinomas tested had a slightly lower beta-pol level than adjacent normal tissue. This study reveals that the base excision repair enzyme DNA polymerase beta is up-regulated in some types of adenocarcinomas and cell lines, but not in others.     

Elevated furin expression in aggressive human head and neck tumors and tumor cell lines

Pro-protein convertases (PCs) are proteases that recognize and cleave precursor proteins. Furin, a well-studied PC, is ubiquitously expressed, and it has been implicated in many physiological and pathological processes. Some substrates for furin, such as membrane type 1 (MT1) matrix metalloproteinase (MMP), an MMP that activates gelatinase, a collagen-degrading enzyme, are associated with the advanced malignant phenotype. This report examines the expression of furin in carcinoma cell lines of different invasive ability. The levels of furin mRNA and protein correlated with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. Furin expression also was investigated in primary head and neck squamous cell carcinomas (HNSCCs). Furin mRNA was not detected in nonmetastasizing carcinomas. In contrast, furin mRNA was expressed in metastasizing HNSCCs. Immunohistochemistry and Western blot analysis confirmed these results at the protein level. Furin activity was investigated indirectly by evaluating the expression of the pro-form and the processed form of MT1-MMP. Metastasizing HNSCCs showed increased expression of MT1-MMP. Furthermore, pro-MT1-MMP expression was noted in most of the nonmetastasizing HNSCCs analyzed by Western blot, and it was absent in the metastasizing HNSCCs. This finding suggests a lower level of furin-mediated MT1-MMP activation in the less aggressive cancers. These observations indicate that furin plays a role in tumor progression. Its overexpression in more aggressive or metastasizing cancers resulted in increased MMP processing.     

Extragonadal and pediatric germ cell tumors.

This article presents a review of the spectrum of extragonadal germ cell tumors, a fascinating group of rare and biologically diverse tumors. Although like testicular germ cell tumors, these tumors are chemotherapeutically responsive, the overall prognosis is not as good. Pediatric germ cell tumors share many of the biologic characteristics of the adult tumors but are more likely to be benign. An unusual aspect of mediastinal germ cell tumors, in particular, is their association with nontreatment-related hematologic malignancies.     

Effect of sodium butyrate on pro-matrix metalloproteinase-9 and -2 differential secretion in pediatric tumors and cell lines

Matrix metalloproteinases (MMPs) are enzymes responsible for extracellular matrix degradation and contribute to local and distant cell invasion during cancer progression or metastasis. The effects of chromatin structure on gene expression and the use of histone deacetylase inhibitors such as sodium butyrate (NaBu) may directly influence pro-MMPs secretion. In the present study, we evaluated the effect of NaBu on pro-MMP-9 and pro-MMP-2 secretion in human Jurkat and HT1080 cells, and in 36 pediatric solid tumors. Cell lines and samples were exposed to 8 mM of NaBu and proteinase activity was evaluated in the supernatant by gelatin zymograms. Our results showed, for Jurkat cells treated with NaBu, increases of 2-fold and 1.5-fold in pro-MMP-9 and pro-MMP-2 secretion, respectively. A 50% decrease in pro-MMP-9 secretion due to NaBu was observed in HT1080 cells. NaBu induced a 0.62 reduction in levels of pro-MMP-9 secretion in untreated tumors. For cell lines and some NaBu-treated tumors we found histone H4 hyperacetylation. We conclude that pro-MMPs gene expression and their secretion can be epigenetically mis-regulated in tumoral processes.     

Selective cancer-germline gene expression in pediatric brain tumors

Cancer-germline genes (CGGs) code for immunogenic antigens that are present in various human tumors and can be targeted by immunotherapy. Their expression has been studied in a wide range of human tumors in adults. We measured the expression of 12 CGGs in pediatric brain tumors, to identify targets for therapeutic cancer vaccines. Real Time PCR was used to quantify the expression of genes MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, NY-ESO-1 and GAGE-1,2,8 in 50 pediatric brain tumors of different histological subtypes. Protein expression was examined with immunohistochemistry. Fifty-five percent of the medulloblastomas (n = 11), 86% of the ependymomas (n = 7), 40% of the choroid plexus tumors (n = 5) and 67% of astrocytic tumors (n = 27) expressed one or more CGGs. Immunohistochemical analysis confirmed qPCR results. With exception of a minority of tumors, the overall level of CGG expression in pediatric brain tumors was low. We observed a high expression of at least one CGG in 32% of the samples. CGG-encoded antigens are therefore suitable targets in a very selected group of pediatric patients with a brain tumor. Interestingly, glioblastomas from adult patients expressed CGGs more often and at significantly higher levels compared to pediatric glioblastomas. This observation is in line with the notion that pediatric and adult glioblastomas develop along different genetic pathways.     

hMLH1 and hMSH2 expression and BAX frameshift mutations in ovarian cancer cell lines and tumors

The expression of mismatch repair proteins hMSH2 and hMLH1 was investigated in human ovarian cancer cell lines and in biopsies of ovarian carcinomas obtained from 20 patients undergoing surgical operation. By Western blotting analysis hMSH2 protein was detected in all the tumor samples analyzed and in eight out of nine human ovarian cancer cell lines, while hMLH1 was undetectable in four out of 20 ovarian tumors and in five out of nine human ovarian cancer cell lines analyzed. The possible presence of frameshift mutations in the BAX gene, which contains a sequence of eight contiguous guanines in its third exon, was tested in all the samples. All the cell lines presented the normal alleles for the BAX gene while only in one of the tumor samples a heterozygous frameshift mutation was found. The frameshift mutation was associated to a low, almost undetectable, level of BAX protein which was instead present at much higher levels in all the other samples investigated. The results indicate that frameshift mutations in the BAX gene, possibly arising as a consequence of microsatellite instability (detectable in these tumors), is detectable in human ovarian cancer although quantitatively it does not appear to be a major determinant of the low apoptotic response to chemotherapy observed in ovarian cancer cells.      

Erucylphosphocholine-induced apoptosis in chemoresistant glioblastoma cell lines: involvement of caspase activation and mitochondrial alterations

Intrinsic chemoresistance constitutes a major problem in the therapy of malignant gliomas. In vitro experiments with four astrocytoma/glioblastoma (AC/GBM) cell lines revealed that the chemoresistance of two cell lines, A172 and T98G, to cisplatin and etoposide was due to resistance to drug-induced apoptosis. In contrast, all the AC/GBM cell lines tested were sensitive to treatment with the lipophilic ether lipid erucylphosphocholine, ErPC. ErPC-induced apoptosis was independent of wild-type p53-signaling and triggering of the CD95/CD95 ligand (CD95L) system. Inhibition of protein and RNA synthesis by cycloheximide and actinomycin D did not abrogate ErPC-induced apoptosis. However, expression of members of the bcl-2 protein family was modulated during ErPC treatment. Activation of caspase 3 and mitochondrial alterations were central to ErPC-induced apoptosis. We conclude that ErPC-induced activation of the mitochondrial pathway enables cell death in the chemoresistant AC/GBM cells.     

Similarities in glycosylation of human neuroblastoma tumors and cell lines

The presence of fucosyl residues linked alpha 1----3(4) to N-acetylglucosamine was demonstrated on the oligosaccharides from glycoproteins of 11 human neuroblastoma tumors from ten different patients. This finding is in complete agreement with the previous report that human neuroblastoma cell lines contained an unusually large proportion of metabolically incorporated L-[3H]fucose in this specific linkage (U. V. Santer and M. C. Glick, Cancer Res., 43:4159-4166, 1983). Furthermore, the glycopeptides derived from the neuroblastoma tumors had a low percentage of fucose-containing biantennary oligosaccharides as determined by affinity to concanavalin A-Sepharose and in this characteristic were similar to glycopeptides from virus transformed and other tumor cells. To obtain these results, the tumor cells were labeled metabolically for 48 h with L-[3H]fucose. The cells were harvested and digested with Pronase, and the glycopeptides were isolated and treated with alpha-L-fucosidase from almonds, specific for the release of fucose linked alpha 1----3(4) to N-acetylglucosamine. A portion of the glycopeptides was characterized by serial affinity chromatography on immobilized concanavalin A and lentil lectin. The phenotypic similarity of the tumor cells to the cell lines, particularly CHP-134, included the paucity of biantennary oligosaccharides and the presence of fucosyl residues on the multiantennae of the glycopeptides.     

Genome-wide gene copy number and expression analysis of primary gastric tumors and gastric cancer cell lines

Background  

Gastric cancer is one of the most common malignancies worldwide and the second most common cause of cancer related death. Gene copy number alterations play an important role in the development of gastric cancer and a change in gene copy number is one of the main mechanisms for a cancer cell to control the expression of potential oncogenes and tumor suppressor genes.     

HER-2 receptor expression, localization, and activation in colorectal cancer cell lines and human tumors

The HER-2/neu oncogene encodes a 185 kD protein that is phosphorylated upon ligand binding to other HER/erbB members and regulates cell growth and differentiation. Given that HER-2 receptor blockade can inhibit the growth of colon cancer cell lines and tumor xenografts, we investigated the frequency, localization and phosphorylation status of HER-2 in colon cancer cell lines and in human tumors. Protein expression was analyzed in relation to mRNA levels, HER-2 amplification, and clinicopathological variables. Colon cancer cell lines constitutively expressed HER-2 proteins and none showed HER-2 amplification by fluorescence in situ hybridization. Cell fractionation and immunoblotting showed HER-2 in both the membrane and cytosolic compartments. Primary colorectal carcinomas (n = 96) and their metastases (n = 25) were examined by immunohistochemistry. Strong membrane HER-2 staining was detected in 5 (5%) of primaries and in 3 (12%) metastases (p = 0.36). Membrane but not cytoplasmic localization was strongly associated with HER-2 gene amplification (p = 0.007). Cytoplasmic HER-2 staining was found in 61 (63.5%) of primary tumors and localization was confirmed by immunoelectron microscopy that also showed plasma membrane HER-2. Using real-time quantitative RT-PCR, HER-2 mRNA was increased in tumors with membrane compared to cytoplasmic staining (r = 0.66, p = 0.001). Cytoplasmic HER-2 was associated with tumor differentiation (p = 0.018), but not other clinicopathological variables. By immunoblotting, heterogeneity was seen in HER-2 levels with downregulation in 4 of 7 tumors relative to normal epithelia that uniformly expressed HER-2. Phosphorylated HER-2 was detected in approximately 50% of tumors and in normal mucosa. In conclusion, HER-2 is expressed constitutively in colon cancer cell lines and demonstrates relatively distinct localization patterns in human tumors. Strong membrane immunoreactivity is associated with high levels of HER-2 mRNA and gene amplification whereas cytoplasmic HER-2 is detected frequently and seems to be a marker of tumor differentiation.