Gene dosage, expression, and ontology analysis identifies driver genes in the carcinogenesis and chemoradioresistance of cervical cancer.

Integrative analysis of gene dosage, expression, and ontology (GO) data was performed to discover driver genes in the carcinogenesis and chemoradioresistance of cervical cancers. Gene dosage and expression profiles of 102 locally advanced cervical cancers were generated by microarray techniques. Fifty-two of these patients were also analyzed with the Illumina expression method to confirm the gene expression results. An independent cohort of 41 patients was used for validation of gene expressions associated with clinical outcome. Statistical analysis identified 29 recurrent gains and losses and 3 losses (on 3p, 13q, 21q) associated with poor outcome after chemoradiotherapy. The intratumor heterogeneity, assessed from the gene dosage profiles, was low for these alterations, showing that they had emerged prior to many other alterations and probably were early events in carcinogenesis. Integration of the alterations with gene expression and GO data identified genes that were regulated by the alterations and revealed five biological processes that were significantly overrepresented among the affected genes: apoptosis, metabolism, macromolecule localization, translation, and transcription. Four genes on 3p (RYBP, GBE1) and 13q (FAM48A, MED4) correlated with outcome at both the gene dosage and expression level and were satisfactorily validated in the independent cohort. These integrated analyses yielded 57 candidate drivers of 24 genetic events, including novel loci responsible for chemoradioresistance. Further mapping of the connections among genetic events, drivers, and biological processes suggested that each individual event stimulates specific processes in carcinogenesis through the coordinated control of multiple genes. The present results may provide novel therapeutic opportunities of both early and advanced stage cervical cancers.

Expression of 14-3-3sigma in cervical squamous cell carcinomas: relationship with clinical outcome.

14-3-3 sigma (sigma) sequesters the cdc2-cyclin B1 complex in the cytoplasm resulting in G2 arrest. Inactivation and reduced expression of 14-3-3sigma have been reported in a varity of cancers. In the present study, we investigated the expression of 14-3-3sigma in a series of 297 cervical squamous cell carcinoma (SCC) to clarify the prognostic value. Using immunohistochemical methods we found high levels of 14-3-3sigma protein in cytoplasm of 143 (48.1%), in nucleus of 113 (38.0%) and in both cytoplasm and nucleus of 147 (49.5%) cases, whereas, low levels were present in cytoplasm of 154 (51.9%), in nucleus of 184 (62.0%) and in both cytoplasm and nucleus of 150 (50.5%) cases. Levels of 14-3-3sigma mRNA measured by reverse-transcription polymerase chain reaction (RT-PCR) and 14-3-3sigma protein were not significant associated. 14-3-3sigma expression in cytoplasm, nuclear and cytoplasm/nuclear were not significantly correlated to disease-specific survival or disease-free survival. In conclusion, reduced expression of 14-3-3sigma protein in the cytoplasm and shuttle of 14-3-3sigma protein into the nucleus in a relatively high number of cases indicate that 14-3-3sigma may be important in the carcinogenesis of cervical SCCs by two different mechanisms; reduction and nuclear translocation of 14-3-3sigma protein. Furthermore, the non-significant correlation between expression levels of 14-3-3sigma mRNA and protein support a post-transcriptional regulation in cervical SCCs. The protein has no prognostic value in cervical cancers.

Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis.

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC-->TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC-->TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.

Genome-wide estimation of transcript concentrations from spotted cDNA microarray data.

A method providing absolute transcript concentrations from spotted microarray intensity data is presented. Number of transcripts per microg total RNA, mRNA or per cell, are obtained for each gene, enabling comparisons of transcript levels within and between tissues. The method is based on Bayesian statistical modelling incorporating available information about the experiment from target preparation to image analysis, leading to realistically large confidence intervals for estimated concentrations. The method was validated in experiments using transcripts at known concentrations, showing accuracy and reproducibility of estimated concentrations, which were also in excellent agreement with results from quantitative real-time PCR. We determined the concentration for 10,157 genes in cervix cancers and a pool of cancer cell lines and found values in the range of 10(5)-10(10) transcripts per microg total RNA. The precision of our estimates was sufficiently high to detect significant concentration differences between two tumours and between different genes within the same tumour, comparisons that are not possible with standard intensity ratios. Our method can be used to explore the regulation of pathways and to develop individualized therapies, based on absolute transcript concentrations. It can be applied broadly, facilitating the construction of the transcriptome, continuously updating it by integrating future data.

Gene expressions and copy numbers associated with metastatic phenotypes of uterine cervical cancer.

BACKGROUND: A better understanding of the development of metastatic disease and the identification of molecular markers for cancer spread would be useful for the design of improved treatment strategies. This study was conducted to identify gene expressions associated with metastatic phenotypes of locally advanced cervical carcinomas and investigate whether gains or losses of these genes could play a role in regulation of the transcripts. Gene expressions and copy number changes were determined in primary tumors from 29 patients with and 19 without diagnosed lymph node metastases by use of cDNA and genomic microarray techniques, respectively. RESULTS: Thirty-one genes that differed in expression between the node positive and negative tumors were identified. Expressions of eight of these genes (MRPL11, CKS2, PDK2, MRPS23, MSN, TBX3, KLF3, LSM3) correlated with progression free survival in univariate analysis and were therefore more strongly associated with metastatic phenotypes than the others. Immunohistochemistry data of CKS2 and MSN showed similar relationships to survival. The prognostic genes clustered into two groups, suggesting two major metastatic phenotypes. One group was associated with rapid proliferation, oxidative phosphorylation, invasiveness, and tumor size (MRPS23, MRPL11, CKS2, LSM3, TBX3, MSN) and another with hypoxia tolerance, anaerobic metabolism, and high lactate content (PDK2, KLF3). Multivariate analysis identified tumor volume and PDK2 expression as independent prognostic variables. Gene copy number changes of the differentially expressed genes were not frequent, but correlated with the expression level for seven genes, including MRPS23, MSN, and LSM3. CONCLUSION: Gene expressions associated with known metastatic phenotypes of cervical cancers were identified. Our findings may indicate molecular mechanisms underlying development of these phenotypes and be useful as markers of cancer spread. Gains or losses of the genes may be involved in development of the metastatic phenotypes in some cases, but other mechanisms for transcriptional regulation are probably important in the majority of tumors.

Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery.

BACKGROUND: Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure. RESULTS: 5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G2(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C). CONCLUSION: Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies.

Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction.

BACKGROUND: High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure. RESULTS: All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range. CONCLUSIONS: It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data.

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.

Msh2 deficiency increases susceptibility to benzo[a]pyrene-induced lymphomagenesis.

DNA mismatch repair (MMR) is essential for repair of single-base mismatches and insertion/deletion loops. MMR proteins also participate in cellular response to DNA damaging agents such as various alkylating agents. Mice deficient in the MMR gene Msh2 develop tumors earlier after exposure to alkylating agents when compared to unexposed mice. The interaction between the MMR system and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has not been investigated in vivo. Here, we show that treatment of Msh2-deficient mice with B[a]P enhances susceptibility to lymphomagenesis. Carrying at least one intact copy of the Msh2 gene had a protective effect. B[a]P treatment only induced lymphomas in 3 of the 40 (7.5%) mice with at least one intact copy of the Msh2 gene as compared to 13 of the 17 (76.5%) Msh2-deficient mice and occurs only after a much longer time period. The B[a]P-DNA adduct levels measured in lung, liver, spleen and forestomach of B[a]P-treated Msh2-/- mice were not significantly different from B[a]P-treated Msh2+/+ mice. In summary, the results suggest that B[a]P accelerates lymphomagenesis in Msh2-deficient mice. Furthermore, Msh2 deficiency does not have any significant effect on B[a]P-DNA adduct levels.

Intratumor chromosomal heterogeneity in advanced carcinomas of the uterine cervix.

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2-4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14-q25 (60% of 10 cases with this aberration), and gain of 2p22-pter (50% of 6 cases), 11qcen-q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and -X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages.