SATB2 enhances migration and invasion in osteosarcoma by regulating genes involved in cytoskeletal organization.

Osteosarcoma (OS) is the most common malignant bone tumor and the majority of recurrences are due to metastasis. However, the molecular mechanisms that regulate OS metastatic spread are largely unknown. In this study, we report that special AT-rich-binding protein 2 (SATB2) is highly expressed in OS cells and tumors. Short hairpin RNA-mediated knockdown of SATB2 (sh-SATB2) decreases migration and invasion of OS cells without affecting proliferation or viability. Microarray analysis identified genes that were differentially regulated by SATB2 including the actin-binding protein Epithelial Protein Lost In Neoplasm (EPLIN), which was upregulated in sh-SATB2 cells. Silencing EPLIN rescues the decreased invasion observed in sh-SATB2 cells. Pathway analyses of SATB2-regulated genes revealed enrichment of those involved in cytoskeleton dynamics, and increased stress fiber formation was detected in cells with SATB2 knockdown. Furthermore, sh-SATB2 cells exhibit increased RhoA, decreased Rac1 and increased phosphorylation of focal adhesion kinase (FAK) and paxillin. These findings identify SATB2 as a novel regulator of OS invasion, in part via effects on EPLIN and the cytoskeleton.

Defining a test for HER-2/neu evaluation in breast cancer in the diagnostic setting.

In breast cancer amplification of the HER-2/neu oncogene and over-expression of the protein product is associated with poor prognosis, predicts response to some chemotherapeutic regimens and is the target for Herceptin treatment. To date there are several methods to assess the amplification/over-expression of HER-2/neu with each having advantages and disadvantages. We have studied amplification and over-expression of HER-2/neu in 250 consecutive cases of breast cancer (220 invasive and 30 in situ carcinomas) presenting to the Department of Pathology at Women's College Campus of Sunnybrook and Women's College Health Sciences Center. Thirty percent of the invasive carcinomas were node positive. HER-2/neu protein over-expression was assessed by immunohistochemistry (IH) using antibody CB11 and amplification of the gene by differential PCR. The percentage of tumor cells showing CB11 staining was determined and the most significant cut off point for positivity was > or =10% moderate or strong complete membranous staining. The gene was considered amplified if the density score of the product was > or =2. There was 94% concordance between the two methods (P value.0001). Both methods were positive in 16% of cases and negative in 78% of cases. Discrepant cases were examined by FISH which confirmed the IH results in 9/11 invasive carcinomas. These results show that there is excellent concordance between IH and PCR. However, immunohistochemistry is easier to perform and cheaper than PCR and could be used in routine assessment of HER-2/neu in breast cancer patients.

Identification of a novel zinc finger gene, zf5-3, as a potential mediator of neuroblastoma differentiation.

We established a unique parental neuroblastoma cell line, NUB-7, which mimics the bipotentiality of neuroblastoma in vivo along neuronal and Schwann cell lineages following dibutyryl cAMP and retinoic acid treatments, respectively. Differential display identified a putative novel zinc finger gene as a potential differentiation-responsive gene coincident with retinoic acid treatment of NUB-7. This cDNA clone, now designated zf5-3, was mapped to chromosome 19 using somatic cell hybrids, and a larger cDNA clone further localized this gene to band 13.1-13.2 by fluorescent in situ hybridization. zf5-3 possesses 4 characteristic zinc finger DNA-binding motifs as determined by its nucleic acid and proposed amino acid sequence. Expression of zf5-3 is restricted to fetal neuronal, hepatic and renal tissues and their tumor-derived cell lines, including 8/9 neuroblastomas and 2/2 malignant rhabdoid tumors of kidney. The restricted expression in the kidney of zf5-3 to collecting tubules and ureter epithelium is suggestive of an ectodermal histogenesis of malignant rhabdoid tumors of kidney. During development of the fetal human brain, high levels of zf5-3 mRNA are restricted to the mitotically active, undifferentiated neuroblasts. Morphological evidence of overt differentiation was generally accompanied by a marked loss in zf5-3 expression. Therefore, the neuronal tissue expression profile and the down-regulation coincident with retinoic acid-induced neuroblastoma maturation implicate zf5-3 as a potential mediator of their differentiation.

Chromosomal localization of phospholipase A2 activating protein, an Ets2 target gene, to 9p21.

A murine Ets2 target gene isolated by differential display cloning was identified as the phospholipase A2 activating protein (PLAA) gene. A 2.7-kb human cDNA demonstrating high homology to mouse and rat Plaa genes was then isolated and characterized. Human PLAA contains six WD-40 repeat motifs and three different protein kinase consensus domains. Fluorescence in situ hybridization (FISH) mapping placed PLAA on chromosome 9p21, a region frequently deleted in various cancers. A comprehensive mapping strategy was employed to define further the chromosomal localization of PLAA relative to CDKN2A within the 9p21 locus. Radiation hybrid mapping placed the gene 7.69 cR from WI-5735 (LOD >3.0), a marker in close proximity to CDKN2A and CDKN2B. Yeast artificial chromosome (YAC) mapping localized PLAA proximal to the CDKN2A/CDKN2B genes and to a region flanked by D9S171 and INFA commonly deleted in many neoplasms. Two YACs contained both PLAA and D9S259, a marker present in a second more proximal minimal deleted region observed in cutaneous melanoma and squamous cell lung carcinoma. Double-color fiber FISH mapping confirmed the location of PLAA centromeric to D9S171 and CDKN2A/CDKN2B. The mapping data suggest a possible tumor suppressor role for this gene.

Detection of squirrel monkey retroviral sequences in interferon samples.

BACKGROUND/AIMS: Interferons have been used therapeutically in viral infections, and as immunomodulants in the treatment of different types of cancers. Interferons have been prepared from human lymphoid cell-lines, such as Namalwa, that contain integrated copies of squirrel monkey retrovirus proviral DNA. Squirrel monkey retrovirus is related to simian type D retroviruses, such as Mason-Pfizer monkey virus. Thus it is important to determine if these retroviral sequences are present in interferon preparations purified from human cell lines. METHODS: DNA samples were prepared from 75 commercial interferon preparations and analyzed for squirrel monkey retrovirus sequences by polymerase chain reaction and DNA sequencing. Since single polymerase chain reaction is not as sensitive, a nested polymerase chain reaction strategy was devised in order to detect squirrel monkey retrovirus-pol sequences. Amplification of beta-actin (human) sequences was used to confirm that samples contained human genomic DNA. To determine the authenticity of squirrel monkey retrovirus sequences, we analyzed amplified products by Southern blot hybridization and direct DNA sequencing. RESULTS/CONCLUSIONS: Thirty-nine samples were positive for squirrel monkey retrovirus-pol sequences by nested polymerase chain reaction. It is noteworthy that 29 samples were either weakly or very weakly positive by single polymerase chain reaction, thus stressing the importance of our sensitive polymerase chain reaction assay. However, it remains to be determined whether the residual DNA sequences detected by our sensitive nested polymerase chain reaction assay have biological consequences.

Breast cancer genome anatomy: correlation of morphological changes in breast carcinomas with expression of the novel gene product Di12.

To determine which genes may be activated or inactivated during breast cancer development, we employed two cloning strategies (subtractive hybridization and differential display) using RNA samples from a human breast tumor and its matching normal breast cell line. Of 950 clones isolated, 102 cDNA inserts were analysed by DNA sequencing and database searching. We found 30 clones that were obviously unidentified, with no significant homology to any listed human gene. We focused upon one of the novel genes, Di12, that is differentially expressed as a 1.35 kb RNA in breast cancer tissues and cell-lines, and in several normal tissues. A full length cDNA of this gene was cloned, and its DNA sequence revealed an open reading frame of 339 amino acids. Antibodies to the ten N-terminal amino acids were developed to investigate the expression of Di12 in breast cancer cell-lines and tumors. The Di12 protein was found in tissue sections of infiltrating ductal carcinomas (IDCs), but not in benign or normal breast specimens. RT-PCR analysis confirmed expression of Di12 in 80% of infiltrating ductal carcinomas (IDCs). As IDC constitutes approximately 70% of breast cancers seen clinically, the level of Di12 expression may be predictive of disease progression.

Bacterial mutagenicity of dithiocarbamate fungicide thiram.

In the present work, within a project of re-evaluation of authorized pesticides coordinated by PZH (National Institute of Hygiene) we aimed at looking for a mechanism of induction of chromosomal aberrations by thiram. We checked its ability to damage bacterial DNA.

Selection of probes for fluorescence in situ hybridization analysis by differential display polymerase chain reaction of mRNA from rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a malignancy of skeletal muscle derivation encompassing two major subtypes, embryonal and alveolar, which differ in clinical behavior and genetic markers. Because RMS is a relatively circumscribed tumor system for which the beginnings of a molecular genetic framework are in place, it becomes an ideal model for the application of improved methods of molecular genetic analysis. We have applied the technique known as differential display polymerase chain reaction (DD-PCR) to characterize expression of RNA in rhabdomyosarcoma subtypes. Our studies have shown that DD-PCR generates a characteristic electrophoretic profile that can be used to isolate subtype specific probes for fluorescence in situ hybridization (FISH) analysis. We have isolated two cDNA fragments and obtained clones suitable for FISH mapping to metaphase chromosomes. One probe was mapped to the centromeric region of human chromosome 22 and the other probe to the human chromosome band 6q25-26. This approach demonstrates the utility of DD-PCR as a technique for isolating novel cDNA expressed in tumors and their subsequent use as probes for FISH analysis. As more genes are identified by DD-PCR and their roles in tumorigenesis become defined, they are likely to provide novel targets for future molecular cytogenetic analysis.

Molecular analysis of mutations induced by ethylating N-nitroso compounds in the lacI gene of Escherichia coli.

We have investigated the mutational specificities of two ethylating agents, N-nitroso-N,N-diethylamine (NDEA) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in the NC+ domain of the lacI gene in Escherichia coli. G:C-->A:T transitions predominated in each of the two spectra, but its importance varies. While 95% of mutations induced by ENNG are G:C-->A:T transition, they account for only 61% of the total events following NDEA treatment. A:T-->G:C transitions are more often recovered following treatment with NDEA than with ENNG. Deletions, duplications, and frameshifts were also recovered after treatment with NDEA, but not ENNG. Mutations obtained in this study are also compared with those induced by corresponding methylating agents. The 5' flanking base appears to affect the distribution of the G:C-->A:T events. G:C-->A:T transition appears more likely to be recovered at 5'-PuG-3' sites. NDEA, however, induced a significant number of G:C-->A:T changes at 5-PuC-3' sites (51%). The possible reasons for these site-specificities are discussed. Although these alkylating agents most likely induce the bulk of their mutation via common mutagenic intermediates, each agent does induce a characteristic spectrum in which specific sites are enhanced or reduced.

Primitive neuroectodermal tumors of the cerebrum and cerebellum: absence of t(11;22) translocation by RT-PCR analysis.

Cytogenetic analysis of peripheral primitive neuroectodermal tumors (PNETs) has demonstrated a consistent primary chromosomal change characterized by a reciprocal translocation t(11;22)(q24:q12). In the central nervous system PNETs, most frequent of which are the cerebellar medulloblastomas, the most prevalent chromosomal abnormalities include deletions and unbalanced translocations. The recent cloning of the t(11;22) breakpoint has revealed the fusion of the human FLI-1 gene on chromosome 11q24 with a gene EWS on chromosome 22q12 and permitted detection of fusion transcripts. Molecular genetic analysis for the presence of EWS/FLI-1 fusion transcripts by the reverse transcriptase-polymerase chain reaction has recently been applied to peripheral PNETs. In the present study, we analyzed eight central PNETs by reverse transcriptase-polymerase chain reaction for EWS/FLI-1 fusion transcripts. The tumors included six PNETs of the cerebellum, one supratentorial PNET of the frontal lobe and one PNET of the pineal region. Polymerase chain reaction analysis in all eight cases failed to reveal a t(11;22) translocation indicating that this is not a cytogenetic abnormality of the central PNETs. Reverse transcriptase-polymerase chain reaction analysis of EWS/FLI-1 fusion transcripts provides a novel adjunctive tool in the differentiation of central versus peripheral PNET.

Large-scale mutational analysis of EMS-induced mutation in the lacI gene of Escherichia coli.

Mutational spectra produced by mutagens in various repair backgrounds can provide important information about the roles of different repair systems in the mutagenic process. Until recently, such studies have been restricted to the characterisation of comparatively small numbers of mutants or reversion analysis at relatively few sites. The colony hybridisation method used in this study in conjunction with DNA sequencing allows the characterisation of large numbers of mutants and therefore allows analysis of resultant mutational distributions to be made with confidence. We have determined the DNA alterations recovered after treatment with EMS in the N-terminal region of the lacI gene of E. coli. A total of 1138 and 1102 independent lacI-d mutants were characterised in Uvr+ and UvrB-, respectively. Consistent with the known ethylating ability of this compound, the predominant mutation was G:C-->A:T transitions, which accounted for 97% and 93% in Uvr+ and UvrB- strains, respectively. An analysis of the DNA context of mutation induction indicates differential reparability by the Uvr repair pathway. Excision repair appears to more efficiently counter EMS-induced G:C-->A:T transitions at sites flanked by A:T base pairs. However, the influence of excision repair on the ultimate distribution of mutation can not be easily defined with respect to neighbouring sequence.

Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli. VI: Analysis of methylene chloride-induced mutational distribution in Uvr+ and UvrB- strains.

To better understand the mechanisms of mutagenesis by the carcinogen, methylene chloride (DCM), we have determined the nature and distribution of forward mutations induced by DCM in the N-terminal region of the lacI gene of Escherichia coli. A total of 116 lacI-d mutations (50 from Uvr+, 66 from UvrB- strain) were characterized by DNA sequencing. Both similarities and differences were observed. Although in both strains base substitutions predominated (74-88%) the distribution among the classes differed. In the case of the Uvr+ strain, DCM substantially increased the frequency of G:C-->C:G transversion and duplication events. Direct repeats were not observed at the endpoints of the duplications, however, all endpoints were in an A:T-rich region. In contrast, in the UvrB- strain, DCM induced A:T-->G:C, A:T-->C:G, G:C-->C:G events as well as deletions. The mutational spectra presented here represent a first step in the elucidation of the mechanism(s) of DCM-induced mutation.

Genotoxic effects of thiram evaluated by sister-chromatid exchanges in human lymphocytes.

The purpose of this investigation was to study the genotoxic potential of thiram (CAS No. 137-26-8) using an in vitro sister-chromatid exchange (SCE) assay with human lymphocytes. The results indicate that thiram and its metabolites increase the SCE frequencies 2-fold over those observed in the negative controls. The standard inducers cyclophosphamide and ethyl methanesulfonate increased SCE frequencies 10- and 4-fold, respectively, over untreated levels.