Supplementary Table S3 from Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy

Supplementary Table S5 from Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy

Supplementary Table S4 from Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy

Supplementary Table S2 from Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy

Cytogenetic aberration and loss of heterozygosity (LOH) are documented on chromosome 6 in many cancers and the introduction of a neo-tagged chromosome 6 into breast cancer cell lines mediates suppression of tumorigenicity. In this study, we described the identification of KIAA1949 (phostensin) as a putative tumor suppressor gene. Our microarray analysis screened 25,985 cDNAs between a tumorigenic and metastatic breast cancer cell line MDA-MB-231 and the chromosome 6-mediated suppressed, non-tumorigenic and non-metastatic derivative cell line MDA/H6, resulting in the identification of 651 differentially expressed genes. Using customized microarrays containing these 651 cDNAs and 117 controls, we identified 200 frequently dysregulated genes in 10 breast cancer cell lines and 5 tumor tissues using MDA/H6 as reference. Our bioinformatics analysis revealed that chromosome 6 encodes 25 of these 200 genes, with 4 downregulation and 21 upergulation. Northern analysis validated microarray results and was used to detect the size and number of RNA species of 2 downregulated (KIAA1949, PTK7) and 2 upregulated (SFRS3, HMGN3) genes in the cell lines. Particularly, the KIAA1949 gene at 6p21.33, a band region with the frequent cytogenetic aberration and LOH encodes 4 poly(A)-RNA species (4.6-, 4-, 3- and 1.5-kb) in multiple normal and breast cancer samples. Microarray analysis revealed KIAA1949 downregulation in 86.7% (n=15) of breast cancer cell lines and tumor tissues. Northern analysis demonstrated undetectable and decreased expression of KIAA1949 in 100% (n=10) of breast cancer cell lines. Taken together, these results strongly suggest KIAA1949 is a novel candidate breast cancer suppressor gene.

AbstractHuman malignant melanoma has poor prognosis because of resistance to apoptosis and therapy. We describe identification of the expression profile of 1,037 mitochondria-focused genes and 84 survival-apoptosis genes in 21 malignant melanoma cell lines and 3 normal melanocyte controls using recently developed hMitChip3 cDNA microarrays. Unsupervised hierarchical clustering analysis of 1,037 informative genes, and 84 survival-apoptosis genes, classified these malignant melanoma cell lines into type A (n = 12) and type B (n = 9). Three hundred fifty-five of 1,037 (34.2%) genes displayed significant (P ≤ 0.030; false discovery rate ≤ 3.68%) differences (±≥2.0-fold) in average expression, with 197 genes higher and 158 genes lower in type A than in type B. Of 84 genes with known survival-apoptosis functions, 38 (45.2%) displayed the significant (P < 0.001; false discovery rate < 0.15%) difference. Antiapoptotic (BCL2, BCL2A1, PPARD, and RAF1), antioxidant (MT3, PRDX5, PRDX3, GPX4, GLRX2, and GSR), and proapoptotic (BAD, BNIP1, APAF1, BNIP3L, CASP7, CYCS, CASP1, and VDAC1) genes expressed at higher levels in type A than in type B, whereas the different set of antiapoptotic (PSEN1, PPP2CA, API5, PPP2R1B, PPP2R1A, and FIS1), antioxidant (HSPD1, GSS, SOD1, ATOX1, and CAT), and proapoptotic (ENDOG, BAK1, CASP2, CASP4, PDCD5, HTRA2, SEPT4, TNFSF10, and PRODH) genes expressed at lower levels in type A than in type B. Microarray data were validated by quantitative reverse transcription-PCR. These results showed the presence of two types of malignant melanoma, each with a specific set of dysregulated survival-apoptosis genes, which may prove useful for development of new molecular targets for therapeutic intervention and novel diagnostic biomarkers for treatment and prognosis of malignant melanoma.[Mol Cancer Ther 2009;8(5):OF1–13]

Posttraumatic stress disorder (PTSD) is associated with decreased activity in the dorsolateral prefrontal cortex (DLPFC), the brain region that regulates working memory and preparation and selection of fear responses. We investigated gene expression profiles in DLPFC Brodmann area (BA) 46 of postmortem patients with (n=6) and without PTSD (n=6) using human mitochondria-focused cDNA microarrays. Our study revealed PTSD-specific expression fingerprints of 800 informative mitochondria-focused genes across all of these 12 BA46 samples, and 119 (+/->1.25, p<0.05) and 42 (+/->1.60, p<0.05) dysregulated genes between the PTSD and control samples. Quantitative RT-PCR validated the microarray results. These fingerprints can essentially distinguish the PTSD DLPFC BA46 brains from controls. Of the 119 dysregulated genes (+/-> or =125%, p<0.05), the highest percentages were associated with mitochondrial dysfunction (4.8%, p=6.61 x 10(-6)), oxidative phosphorylation (3.8%, p=9.04 x 10(-4)), cell survival-apoptosis (25.2%, p<0.05) and neurological diseases (23.5%, p<0.05). Fifty (50) dysregulated genes were present in the molecular networks that are known to be involved in neuronal function-survival and contain 7 targets for neuropsychiatric drugs. Thirty (30) of the dysregulated genes are associated with a number of neuropsychiatric disorders. Our results indicate mitochondrial dysfunction in the PTSD DLPFC BA46 and provide the expression fingerprints that may ultimately serve as biomarkers for PTSD diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of PTSD.

Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines.

Introduction of human chromosome 6 into malignant melanoma cell line UACC903 resulted in generation of the chromosome 6-mediated suppressed cell subline UACC903(+6) that displays attenuated growth rate, anchorage-dependency, and reduced tumorigenicity. We have showed that overexpression of a chromosome 6-encoded tumor suppressor gene led to partial suppression to UACC903 cell growth. We now describe the differences in apoptosis and cell cycle between UACC903 and UACC903(+6) before and after UV irradiation. MTT assay revealed 86.92+/-8.24% of UACC903 cells viable, significantly (p<0.01) higher than 48.76+/-5.31% of UACC903(+6), at 24 hr after 254-nm UV irradiation (40 J/M(2)). Before UV treatment, flow cytometry analysis revealed 6.06+/-0.20% apoptosis in UACC903, significantly (p=0.01) lower than 6.67+/-0.15% in UACC903(+6). The G0/G1, S and G2/M phase cells of UACC903 were, respectively, 54.10+/-0.59%, 22.31+/-0.50% and 16.85+/-0.25%, all significantly (p<0.01) different from the corresponding percentages (58.82+/-0.35%, 20.48+/-0.05%, and 13.17+/-0.45%) of UACC903(+6). After the UV treatment, UACC903 cells in apoptosis, G0/G1, S, and G2/M became 12.59+/-0.17%, 38.90+/-0.67%, 19.74+/-0.70%, and 27.01+/-0.66%, respectively, while UACC903(+6) cells were 24.16+/-0.48%, 37.97+/-0.62%, 19.20+/-0.52%, and 15.69+/-0.14%. TUNEL assay revealed 2.31+/-0.62% apoptosis in UACC903, significantly (p<0.01) lower than 9.60+/-1.14% of UACC903(+6), and a linear and exponential increase of apoptosis, respectively, in response to the UV treatment. These results indicate that UACC903(+6) cells have a greater tendency to undergo apoptosis and are thus much more sensitive to UV irradiation. Our findings further suggest a novel mechanism for chromosome 6-mediated suppression of tumorigenesis and metastasis, i.e., through increased cell death.

Pulmonary neuroendocrine tumors vary dramatically in their malignant behavior. Their classification, based on histological examination, is often difficult. In search of molecular and prognostic markers for these tumors, we used cDNA microarray analysis of human transcripts against reference RNA from a well-characterized immortalized bronchial epithelial cell line, BEAS-2B. Tumor cells were isolated by laser-capture microdissection from primary tumors of 17 typical carcinoids, small cell lung cancers, and large cell neuroendocrine carcinomas. An unsupervised, hierarchical clustering algorithm resulted in a precise classification of each tumor subtype according to the proposed histological classification. Selection of genes, using supervised analysis, resulted in the identification of 198 statistically significant genes (P <.004) that also accurately discriminated between 3 predefined tumor subtypes. Two-by-two comparisons of these genes identified classifier genes that distinguished each tumor subtype from the others. Changes in expression of selected differentially expressed genes for each tumor subtype were internally validated by real-time reverse-transcription polymerase chain reaction. Expression of 2 potential classifier gene products, carboxypeptidase E (CPE) and gamma-glutamyl hydrolase (GGH), was validated by immunohistochemistry and cross-validated on additional archival samples of pulmonary neuroendocrine tumors. Kaplan-Meier survival analysis revealed that immunostaining for CPE was a statistically significant predictor of good prognosis, whereas GGH expression correlated with poor prognosis. Thus, cDNA microarray analysis led to the identification of 2 novel biomarkers that should facilitate molecular diagnosis and further study of pulmonary neuroendocrine tumors.

Recent expression profile analyses revealed that lung adenocarcinomas can be divided into several subgroups with diverse pathological features. Because cellular heterogeneity of tumors can confound these analyses, we used laser capture microdissection and microarray expression analysis to characterize the molecular profiles of lung adenocarcinomas. We found 45 genes delineating smokers and nonsmokers that were located at chromosomal loci frequently altered in non-small cell lung cancers, and 27 genes, which were differentially expressed between survivors and nonsurvivors 5 years after surgery. These results are consistent with the hypothesis that the abnormal expression of genes involved in maintaining the mitotic spindle checkpoint and genomic stability, e.g., hBUB3, hZW10, and APC2, contribute to the molecular pathogenesis and tumor progression of tobacco smoke-induced adenocarcinoma of the lung.