Potential role of Shh-Gli1-BMI1 signaling pathway nexus in glioma chemoresistance.

Chemoresistance is a common hurdle for the proper treatment of gliomas. The role of Shh-Gli1 signaling in glioma progression has been reported. However, its role in glioma chemoresistance has not been well studied yet. In this work, we found that Shh-Gli1 signaling regulates the expression of one stem cell marker, BMI1 (B cell-specific Moloney murine leukemia virus), in glioma. Interestingly, we also demonstrated high expression of MRP1 (multi-drug resistance protein 1) in glioma. MRP1 expression was decreased by BMI1 siRNA and Shh-Gli1 cell signaling specific inhibitor GANT61 in our experiments. GANT61 very efficiently inhibited cell colony growth in glioma cell lines, compared to temozolomide. Moreover, a synergic effect of GANT61 and temozolomide drastically decreased the LD50 of temozolomide in the cell colony experiments. Therefore, our results suggest that there is a potential nexus of Shh-Gli1-BMI1 cell signaling to regulate MRP1 and to promote chemoresistance in glioma. Henceforth, our study opens the possibility of facing new targets, Gli1 and BMI1, for the effective treatment of glioma suppression of chemoresistance with adjuvant therapy of GANT61 and temozolomide.

Detección y aislamiento de células inmaduras neuronales a partir de líneas celulares de tumores del sistema nervioso / Detection and isolation of immature neural cells from tumoral cell lines of the nervous system

Introducción: Hoy se sugiere que la teoria de carcinogénesis establecida en las últimas décadas, según la cual el cáncer se produce por el acúmulo de mutaciones en proto-oncogenes y genes supresores de tumores podría cuestionarse, a favor de una teoría más completa que incluya la participación de las denominadas células stem tumorales. Estas células poseerían la capacidad de iniciar y mantener el tumor, además de su propia capacidad de diferenciación y autorrenovación. Objetivo: Determinar el fenotipo de marcadores de diferenciación y la posible existencia de células inmaduras (tal vez células stem) en las líneas celulares de tumores del sistema nervioso. Material y métodos: Se estudió el nivel de expressión de los genes CD133, nestina, Musashi-1, FAS, NCAM1 y GFAP en 27 líneas celulares de tumores del sistema nervioso mediante RT-PCR semicuantitativa y citometría. La línea de neuroblastoma IMR-32 fue sometida a separación celular mediante SdFFF. Resultados: Hemos podido separar diferentes subclones o células en diferentes estadios de diferenciación en la línea de neuroblastoma IMR-32. La correcta tipificación de estas líneas celulares podría ser relevante para establecer tratamientos quimioterápicos o de terapia génica, específicamente dirigidos contra los subclones celulares más inmaduros, que podrían corresponder (o no) a células stem tumorales (AU)

Introduction: Nowadays it is suggested that the theory of carcinogenesis established along the last decades, according to which cancer is produced by the accumulation of mutations in proto-oncogenes and tumor suppressor genes might be questioned in favor of a more complete theory that includes the participation of the so called tumor stem cells. These cells would represent those in charge of initiating and maintaining the tumor; with their own capacity of differentiation and autorenewal. Objective: To determine the phenotype of differentiation markers and the possible existence of immature cells (maybe stem cells) in cell lines of tumors of the nervous system. Material and methods: The level of expression of CD133, nestine, Musashi-1, FAS, NCAM1 and GFAP genes was studied in 27 cell lines of tumors of the nervous system by semiquantitative RT-PCR and cytometry. The neuroblastoma cell line IMR-32 was subjected to cell separation by SdFFF. Results:We could separate different subclones or cells in different stages of differentiation in the neuroblastoma cell line IMR-32. The correct description of these cells might be relevant to set up chemoterapeutic or gene therapy treatments specifically targeted against the most immature subclones, that might correspond (or not) to tumor stem cells (AU)

Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by sedimentation field flow fractionation.

Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

Silencing of DMBT1 in neuroblastoma cell lines is not due to methylation of CCWGG motifs on its promoter.

DMBT1 is one of the putative suppressor genes on 10q25-qter, which frequently lacks expression in many different kind of tumors, such as glioblastoma, and lung, esophageal and colorectal cancer. However, little is known about the reasons for this lack of expression in neoplasia. In a previous report, our group demonstrated how MC-IXC, a neuroblastoma cell line which lacked DMBT1 expression, restored it after a 5-Aza-2'-deoxycitidine treatment. So, we wondered whether DMBT1 aberrant promoter methylation could be responsible for DMBT1 silencing in several tumor cell lines, in spite of the fact that there is no CpG island near the 5' end of the gene. We studied the possibility that methylation in CCWGG sequences of the DMBT1 promoter (where "W" means "A" or "T") is able to silence the gene, as had previously been reported for TP53 in leukemia. We digested genomic DNA by the methylation sensitive restriction enzyme EcoR II (C|CWGG), and made two PCRs to amplify the three CCWGG domains placed in the 1 kb upstream DMBT1 5' end. After the PCRs, we could not find correlation between methylation in CCWGG domains and DMBT1 lack of expression. A positive regulator of DMBT1 might be silenced by aberrant methylation.

Genetic heterogeneity in supratentorial and infratentorial primitive neuroectodermal tumours of the central nervous system.

AIMS: Medulloblastoma (MB), a kind of infratentorial primitive neuroectodermal tumour (PNET), is the most frequent malignant brain tumour in childhood. In contrast, supratentorial PNET (sPNET) are very infrequent tumours, but they are histologically similar to MB, although they present a worse clinical outcome. We investigated the differences in genetic abnormalities between sPNET and MB. METHODS AND RESULTS: We analysed 20 central PNET (14 MB and six sPNET) by conventional comparative genomic hybridization (CGH) in order to determine whether a different genetic profile for each tumour exists. Isochromosome 17q was detected in four of the 14 MB cases, but not in any sPNET. Gains at 17q and 7 happened more frequently in MB, and those at 1q in sPNET. Losses at chromosome 10 were detected only in MB, while losses at 16p and 19p happened more frequently in sPNET. A new amplification site, on 4q12, was detected in two MB. CONCLUSIONS: Central PNET are a heterogeneous group of tumours from the genetic point of view. The present and previous data, together with further results from larger series, might contribute to the establishment of specific treatments for supratentorial and infratentorial PNET.

Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.

Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.

Gain of chromosome 3 and loss of 13q are frequent alterations in pituitary adenomas.

Genetic changes underlying the tumorigenesis of pituitary adenomas (PA) are poorly characterized. To search for characteristic genomic imbalances involved in PA, we examined 38 cases: 12 hormone-secreting (HS) and 26 non-functioning (NF) PA, by comparative genomic hybridization. The most frequent DNA copy number change in both kinds of tumors was loss of 13q. Gains of chromosomes 3, 7 and 14, 6p, and 20q were more frequent in HSPA than in NFPA. These data indicate that the 13q region may harbor tumor suppressor genes determining the tumorigenesis of PA and gain in chromosome 3 may be related to hormone secretion. These findings provide a basis to search for candidate diagnostic markers of HSPA.

Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells.

The sensitivity of non-isotopic PCR-SSCP was compared to direct sequencing of PTEN exons. DNA from leukocytes derived from healthy donors, and from the glioblastoma cell line LN319 was extracted and mixed in different proportions from 0 to 100%. The LN319 cell line contains a point mutation at codon 15 exon 1 of the PTEN gene. The PCR-SSCP experiments demonstrated mutations in samples containing as little as 10% tumor DNA. In contrast, direct DNA sequencing experiments were less sensitive, requiring 30-70% of tumor DNA in the sample, depending on the DNA strand sequenced. In conclusion, PCR-SSCP, in our hands, is more sensitive than automated sequencing for detecting PTEN point mutations. We recommend to always sequence both strands, and take into account that samples containing less than 30% tumor cells should not only be sequenced, but also studied by PCR-SSCP in order to discriminate false negative results.

Lack of gene amplification as a mechanism of CDK4 activation in human neuroblastoma.

Neuroblastoma is the most frequent solid tumor in childhood. We have analysed 48 neuroblastomas of different stages and degrees of cellular differentiation for CDK4 amplification by a fluorescent differential PCR assay. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. We were not able to detect any case of CDK4 gene amplification in the neuroblastomas. In conclusion, CDK4 activation does not seem to be relevant to the development of neuroblastomas, at least through gene amplification.

Differential polymerase chain reaction: a technical comparison of three methods for the detection of CDK4 gene amplification in glioblastomas.

Among the different techniques used to detect oncogene amplification in tumor DNA, Southern blot and differential PCR have been the most frequently used. We report on a technical comparison of three different methods to detect gene amplification by differential PCR: ethidium bromide staining, silver staining (both after standard differential PCR), and fluorescent differential PCR. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. In total agreement with previous studies carried out by Southern blot and differential PCR by other authors, we were able to detect CDK4 amplification in 3 of the 21 glioblastomas (14%), but only by the fluorescent differential PCR method. In conclusion, fluorescent differential PCR is more sensitive than standard differential PCR for detection oncogene amplification in tumor DNAs.

Mutational analysis of the p16 gene in human neuroblastomas.

Neuroblastoma is one of the most frequent tumors in infancy. We analyzed 26 neuroblastomas, two ganglioneuromas, and a neuroblastoma metastasis for mutations and homozygous deletions of the p16 (or MTS1 or CDKN2) gene by means of the polymerase chain reaction (PCR) in combination with the single-strand conformation polymorphism (SSCP) technique and by multiplex PCR analysis. We detected mobility shifts in the SSCP gels in seven cases in the 3 half of exon 2 (named exon 2C) of the p16 gene. By PCR amplification of this particular region and SacII restriction enzyme digestion, we confirmed that those cases had a known polymorphism at codon 140 of the p16 gene. Neither mutations nor homozygous deletions were detected. Our results confirm those of Beltinger et al. (Cancer Res 55:2053-2055, 1995), which showed no p16 mutations or homozygous deletions in 18 primary neuroblastomas and nine tumor-derived cell lines. We conclude that the common pattern of p16 inactivation by homozygous deletion or mutation does not seem to be relevant to the development of neuroblastomas.

Loss of the p16INK4a and p15INK4b genes, as well as neighboring 9p21 markers, in sporadic melanoma.

Although homozygous deletions of the cyclin-dependent kinase inhibitor 2 gene p16INK4a on 9p21 have been reported frequently in metastatic melanoma cell lines, and intragenic mutations within the p16INK4a gene have been detected in familial melanoma kindreds, specific targeting of this gene in the development of sporadic melanoma in vivo remains controversial. Southern analyses were performed in this study to initially assess the frequency of hemi- or homozygous losses of p16INK4a, as well as its neighboring family member, p15INK4b, and other candidate regions within 9p21, in sporadic melanoma. Overall, 22 of 40 (55%) uncultured sporadic melanoma DNAs were determined to harbor deletions of 1-11 markers/genes located on 9p21. This included 10 tumors (25%; 10 of 40) with homozygous deletions limited to either the p16INK4a gene only (20%; 2 of 10), both the p16INK4a and p15INK4b genes (10%; 1 of 10), another novel 9p21 gene, FB19 (10%; 1 of 10), or all three of these genes plus surrounding markers (60%; 6 of 10). In subsequent single-strand conformation polymorphism and sequencing analyses, intragenic mutations in the p16INK4a gene were also revealed in two (10%; 2 of 21) melanoma DNAs that retained one copy of this locus. By comparison, the frequency of pl6INK4a and p15INK4b homozygous deletions, as well as p16INK4a mutations, in melanoma cell lines (analyzed in parallel) was 2-3-fold higher at 61 (23 of 38) and 24% (9 of 38), respectively. These findings indicate that (a) p16INK4a is inactivated in vivo in over one-fourth (27.5%; 11 of 40) of sporadic melanomas; (b) mutation/deletion of p16INK4a may confer a selective growth advantage in vitro; and (c) other 9p21 tumor suppressor genes could be targeted during the development of melanoma.

Chromosome 17 allelic loss and NF1-GRD mutations do not play a significant role as molecular mechanisms leading to melanoma tumorigenesis.

Allelic loss in human cutaneous melanoma has been detected on chromosomes 1p, 6q, 9p, 10q, and 11q. Chromosome 17 contains important tumor suppressor genes such as p53, NM23, and neurofibromatosis type 1 (NF1), which have been implicated in melanoma tumorigenesis. The role of p53 has already been studied by a number of laboratories, showing contrasting results. In the present study, two restriction fragment length polymorphism (RFLP) probes for the NM23 and NF1 genes, together with five other RFLP and four variable number of tandem repeat chromosome 17 probes, were investigated at the loss of heterozygosity (LOH) level in a Southern blot-based assay. The NF1 gene was also tested for LOH by a polymerase chain reaction (PCR)-based approach in two different experiments, using a dinucleotide repeat polymorphic probe at locus D17S250 (17q11.2-q12), and an Alu probe intragenic to the NF1 gene (17q11.2). A PCR single-strand conformation polymorphism assay was included in the study for mutation detection at the NF1-GTPase-activating protein-related domain (GRD). A total of 68 melanocytic tumors were analyzed. LOH was detected in 9 of 87 informative cases (10%). LEW301 (17p11.2-pcen) presented the highest LOH frequency (22%). NM23 showed LOH in 17% of the informative cases, while NF1 did not show either LOH in the Southern blot- and PCR-based experiments or mutations at the NF1-GRD. These results are in concordance with those of previous smaller studies, but when compared with higher LOH frequencies obtained from other chromosomes, these findings indicate that the LOH values found in our study can most likely be attributed to background effect. Thus, chromosome 17 LOH is likely to play and unimportant role as a genetic event in melanoma tumorigenesis. Nevertheless, NF1 merits further study, since homozygous deletions have been detected at this locus in melanoma cell lines.

Absence of mutation at the GAP-related domain of the neurofibromatosis type 1 gene in sporadic neurofibrosarcomas and other bone and soft tissue sarcomas.

The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of downregulating ras by stimulating ras intrinsic GTPase activity. We tested 44 sarcomas, nine of which corresponded to sporadic neurofibrosarcomas, for mutations at the NF1-GRD by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, finding no mutation in every sample tested. We suggest that inactivation of the NF1-GRD by gene mutation seems not to be an important event in the tumorigenesis of sarcomas.

p53 protein expression in viral warts from patients with epidermodysplasia verruciformis.

The p53 protein is the product of a tumour suppressor gene, which is implicated in many human malignancies. p53 expression was investigated by immunohistochemistry in a series of viral warts (n = 12) from five patients with epidermodysplasia verruciformis (EV), using a monoclonal anti-p53 antibody (DO7). p53 expression was also investigated in a series of common warts (n = 8), flat warts (n = 8), and penile bowenoid papulosis (n = 6) from non-EV patients. Immunostaining was positive in 11 of 12 (92%) EV warts, whereas p53 reactivity was negative in most cases of warts from non-EV patients. Exons 5-8 of the p53 gene were screened by the polymerase chain reaction-single strand conformation polymorphism technique in four EV warts, which were strongly stained for p53, and p53 mutations were not detected. These results suggest an association between p53 accumulation (probably of wild type) and EV warts.

Detection of TP53 gene mutations in human sarcomas.

To determine the frequency and type of TP53 mutations in human sarcomas, we examined exons 5-8 of the TP53 gene in 48 sarcomas using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. Nine tumours had mobility-shifts on SSCP analysis, and sequencing of six of these tumours revealed 10 mutations: one insertion, two deletions and seven point mutations (four transitions and three transversions). Four of these mutations resulted in frame-shifts, one in a truncated protein, four cases in mono-allelic point mutations and one case in an altered splice site. These data show that approximately 20% of sarcomas harbour TP53 gene alterations and illustrate a variety of TP53 gene mutation types.

c-myc oncogene amplification and cytometric DNA ploidy pattern as prognostic factors in musculoskeletal neoplasms.

The relationship between c-myc oncogene amplification in neoplastic cells as determined by means of Southern-blot analysis, and their nuclear DNA content as assessed by combined flow and image cytometry, was investigated in fresh tumor specimens from 33 patients with musculoskeletal neoplasms. Amplification, without rearrangement of the c-myc proto-oncogene, was detected in 4 out of 7 bone sarcomas and in 6 out of 26 soft-tissue sarcomas, but in none of 3 benign giant-cell bone tumors. Among the 10 cases with c-myc amplification, 2 were found to be cytometrically DNA diploid, 2 DNA tetraploid, and 6 DNA aneuploid. Conversely, there were 10 tumors displaying extremely aneuploid DNA patterns without c-myc oncogene amplification. Thus, there was no relationship between c-myc amplification and DNA ploidy; neither did the percentage of S-phase cells, as determined by means of image cytometry, correlate significantly with the occurrence of c-myc amplification. A surprising sex-bias was observed; all 6 cases of c-myc-amplified soft-tissue sarcomas occurred in females, whereas none of the 11 males with such sarcomas showed this amplification. When the clinical follow-up data of the patients were scrutinized, it was found that the DNA ploidy pattern of the neoplastic cell nuclei, in combination with the S-phase values, as well as the occurrence of c-myc amplification, yielded prognostic information, being statistically significant 2 years after the diagnosis.

No association between c-myc amplification and TP53 mutation in sarcoma tumorigenesis.

Oncogenes and tumor suppressor genes coparticipate in sarcoma tumorigenesis. We tested 43 sarcomas for c-myc amplification by Southern blot and molecular hybridization techniques and TP53 mutations by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique and direct sequencing of the PCR products. We found eight tumors with c-myc amplification and five different tumors with TP53 mutations but no tumors harbor both c-myc and TP53 alterations. We suggest that c-myc amplification and TP53 mutations do not seem to coparticipate in the tumorigenesis of sarcomas.