Occurrence of senescence-escaping cells in doxorubicin-induced senescence is enhanced by PD0332991, a cyclin-dependent kinase 4/6 inhibitor, in colon cancer HCT116 cells.

Cancer treatment induces cellular senescence, and it is considered to be one of the factors that determines treatment outcome. Senescence can be efficiently induced in cultured cells by DNA-damaging drugs, including doxorubicin (DOX), cisplatin and etoposide. Cells in senescence cease proliferation; however, it has been demonstrated that colonies that are formed from cells escaping senescence appear in drug-induced senescence; however, the conditions influencing the emergence of such senescence-escaping cells (SECs) remain unclear. The present study aimed to investigate the relevance of the cell cycle phase and colony formation in the DOX-induced senescence of human colon cancer HCT116 cells. After release from serum starvation in the presence of DOX, cells synchronously progressed through the cell cycle and were arrested in the G1 and G2/M phases. The ratio of G1 cells arrested immediately by the treatment of G1 phase cells was positively associated with the number of colony-forming cells. A procedure increasing G1-treated G1-arrested cells enhanced colony formation. Co-treatment of PD0332991 with DOX slowed progression of cells in the G1 phase resulting in enhanced colony formation from the increased G1-treated G1-arrested cells. These results may provide useful insights into understanding the emergence of SECs in drug-induced senescence.

A novel mechanism of EML4-ALK rearrangement mediated by chromothripsis in a patient-derived cell line.

INTRODUCTION: EML4-ALK is a driver oncogene in non-small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. METHODS: An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. RESULTS: We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. CONCLUSION: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis.

Amplification of the ABCB1 region accompanied by a short sequence of 200bp from chromosome 2 in lung cancer cells.

Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7. The sequence was identical in the two sublines. A short sequence of 200bp derived from chromosome 2 was incorporated, suggesting translocation between chromosomes 2 and 7. The copy number of the short sequence was comparable to that of the neighboring sequence, suggesting coamplification. The timing of the occurrence of the putative translocation and the initiation of BFB-cycle-driven amplification during the stepwise selection were determined by using the unique junction sequences specific to these events as indicators. The results demonstrated that the translocation occurred at the step of 100 nmol/L treatment and the BFB cycle initiated in the step of 400 nmol/L-treatment. It is likely that the translocation, preceding amplification by several selection steps, activated ABCB1 gene expression. The diversity in amplification profiles between the two sublines was generated by the separately operating BFB cycles, after an initial break-and-fusion that probably occurred in a single cell.

A signature-based method for indexing cell cycle phase distribution from microarray profiles.

BACKGROUND: The cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate). However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods. RESULTS: We developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets. CONCLUSION: The signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.

PrognoScan: a new database for meta-analysis of the prognostic value of genes.

BACKGROUND: In cancer research, the association between a gene and clinical outcome suggests the underlying etiology of the disease and consequently can motivate further studies. The recent availability of published cancer microarray datasets with clinical annotation provides the opportunity for linking gene expression to prognosis. However, the data are not easy to access and analyze without an effective analysis platform. DESCRIPTION: To take advantage of public resources in full, a database named "PrognoScan" has been developed. This is 1) a large collection of publicly available cancer microarray datasets with clinical annotation, as well as 2) a tool for assessing the biological relationship between gene expression and prognosis. PrognoScan employs the minimum P-value approach for grouping patients for survival analysis that finds the optimal cutpoint in continuous gene expression measurement without prior biological knowledge or assumption and, as a result, enables systematic meta-analysis of multiple datasets. CONCLUSION: PrognoScan provides a powerful platform for evaluating potential tumor markers and therapeutic targets and would accelerate cancer research. The database is publicly accessible at http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html.

17Beta-hydroxysteroid dehydrogenase type 12 in human breast carcinoma: a prognostic factor via potential regulation of fatty acid synthesis.

17beta-Hydroxysteroid dehydrogenase type 12 (17beta-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17beta-HSD12 expression was also reported in breast carcinomas but its functions have remained unknown. In this study, we examined the correlation between mRNA expression profiles determined by microarray analysis and tissue E2 concentrations obtained from 16 postmenopausal breast carcinoma cases. No significant correlations were detected between 17beta-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17beta-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17beta-HSD12 using cell-based studies. Small interfering RNA-mediated knockdown of 17beta-HSD12 in SK-BR-3 (estrogen receptor-negative breast carcinoma cell line) resulted in significant growth inhibition, which was recovered by the addition of VLCFAs such as arachidonic acid. The status of 17beta-HSD12 immunoreactivity was also correlated with adverse clinical outcome in cyclooxygenase 2 (COX2)-positive breast cancer patients but not in COX2-negative patients. Therefore, these findings indicated that 17beta-HSD12 was not necessarily related to intratumoral E2 biosynthesis, at least in human breast carcinoma, but was rather correlated with production of VLCFAs such as arachidonic acid, which may subsequently be metabolized to prostaglandins by COX2 and result in tumor progression of the patients.

Comparison of murine gene expression profiles between spontaneous and radiation-induced myelogenous leukemias: stochastic and probabilistic expression variances in the former vs radiation-specific expression commonalities in the latter.

OBJECTIVE: To elucidate the common characteristics of murine radiation-induced myelogenous leukemias, global gene-chip expression profiles were compared with age-matched steady-state bone marrow tissue profiles and spontaneous myelogenous leukemia profiles. MATERIALS AND METHODS: Six each of C3H/He mice-derived radiation-induced and spontaneously developed myelogenous leukemias were analyzed. Bone marrow cells from five each of 2- and 21-month-old mice were used to subtract nonleukemic information in the analysis. mRNAs from individual mice were analyzed separately using 45,101 gene chips followed by computational biological analysis. RESULTS: First, principal component analysis (PCA) was performed to discriminate the gene expression profiles of individual mice with radiation-induced myelogenous leukemia from those of bone marrow cells from 2- or 21-month-old mice. Discriminant union genes for individual leukemias were then selected, which finally yielded 242 genes, among which six are radiation-related genes including Hus-1, Edf1a2, andVegf-c; 16 are apoptosis/cell-death-related genes, 13 are cell-cycle/cell-growth-related genes, and 50 are suppressor/promoter genes. PCA of these 242 genes consistently enabled the discrimination of the radiation-induced leukemias from the spontaneous leukemias. Second, the other components of the same PCA provided four different eigenvector clusters in an unsupervised manner representing four histopathological findings, with which the differential diagnosis in molecular taxonomy was significant as determined by analysis of variance of the global gene expression profiles. CONCLUSION: Discriminant union genes in radiation-induced myelogenous leukemias against spontaneous myelogenous leukemias and age-matched nonleukemic bone marrow profilings generated by unsupervised computational analysis essentially represent probabilistic biomarkers for radiation-induced myelogenous leukemias, which may contribute to developing a model for risk of secondary carcinogenesis in patients treated by whole-body irradiation.

The complicated copy number alterations in chromosome 7 of a lung cancer cell line is explained by a model based on repeated breakage-fusion-bridge cycles.

The drug-resistant lung cancer cell line PTX250, which has been previously established by exposure to an anti-cancer drug paclitaxel, has an increased copy number in the MDR1/ABCB1 locus region. In addition, the flanking regions also exhibit aberrant copy numbers, making the copy number profile of chromosome 7 complicated. In this study, we tested whether the breakage-fusion-bridge (BFB) cycle model can explain such copy number alterations. An analysis using fluorescence in situ hybridization (FISH) with a painting probe demonstrated that the aberrant chromosome, designated chromosome 7(amp), was derived from an intact chromosome 7. Using high-density comparative genomic hybridization arrays, we examined the copy number profile in detail and divided chromosome 7(amp) into seven segments. Based on copy numbers of each segment, which were determined using interphase- and metaphase-FISH analysis, we constructed a formation model for the complicated copy number alteration. Six-time BFB cycles and the cycle-termination by healing of broken ends were presupposed in the model. Locations and orientations of the segments observed in chromosome 7(amp) agreed well with those predicted from the model. Telomere addition was also cytogenetically confirmed. In all, it could be concluded that the complicated copy number alteration found in chromosome 7(amp) is generated from the intact chromosome 7 by the repeated BFB cycles.

The MDR1/ABCB1 regional amplification in large inverted repeats with asymmetric sequences and microhomologies at the junction sites.

A multidrug-resistant lung cancer cell line PTX250, established by treatment with the anti-cancer drug paclitaxel, has been demonstrated to have an increased copy number in the 7q21.12 region including the MDR1/ABCB1 gene. The amplicon is 2.7 megabases in size, and the copy number increase is 11-fold compared with the parental cell line. Here, we examined the amplicon structure and determined nucleotide sequences at both junctions of the amplicon. Fluorescence in situ hybridization analysis using an MDR1 probe demonstrated a cluster of fluorescent signals at the chromosomal end, suggesting an intra-chromosomal amplification. DNA fragments of both junctions were cloned and sequenced. The distal junction was a head-to-head fusion with a 4-base pair (bp) overlap separated by an asymmetric sequence of 1,265 bp, and the proximal junction was a tail-to-tail fusion with a 2-bp overlap intervened by an asymmetric sequence of 2,203 bp. These results suggest that the amplicon has a large palindromic structure with an asymmetric sequence and has been amplified through the breakage-fusion-bridge cycle. Specific sequences, which might be related to the occurrence of double-strand-breakages, were found at or near the junctions of the amplicon -- an inverted repeat in the distal junction and a highly AT-rich region near the proximal junction.

Aromatase localization in human breast cancer tissues: possible interactions between intratumoral stromal and parenchymal cells.

Aromatase is a key enzyme in intratumoral estrogen production required for the production of estrogens through the conversion of serum androgens in postmenopausal breast cancer patients. There have been, however, controversies regarding the intratumoral localization of aromatase in human breast carcinoma tissues. Therefore, we have first examined the intratumoral localization of aromatase mRNA/protein in 19 breast carcinomas using laser capture microdissection/quantitative reverse transcription-PCR (RT-PCR) and immunohistochemistry. Aromatase mRNA and protein were detected in both intratumoral stromal and parenchymal cells in breast carcinoma tissues. Subsequent microarray expression profiling and clustering analyses, in addition to quantitative RT-PCR studies, showed a significant positive correlation between aromatase and estrogen-related receptor alpha mRNA expression in isolated carcinoma cells. We further examined an interaction between stromal cells isolated from human breast carcinoma tissues and breast carcinoma cell lines using a coculture system to study the biological characteristic of aromatase expression in carcinoma cells. Aromatase mRNA and enzyme activity and 17beta-hydroxysteroid dehydrogenase type 1 mRNA in breast carcinoma cell lines, including MCF-7 and SK-BR-3 cells, were up-regulated in the presence of patient-derived 32N or 74T intratumoral stromal cells. The results from steroid conversion assays were also consistent with the findings above. The results of our study also showed that aromatase inhibitors were more effective in inhibiting aromatization induced by coculture in MCF-7 than that in stromal 32N. The examination of the localization of aromatase and its regulation, including the interactions existing between different cell types in human breast carcinoma tissues, may provide important information as to achieving better clinical response to aromatase inhibitors in breast cancer patients.

Gene amplification and expression in lung cancer cells with acquired paclitaxel resistance.

A paclitaxel-resistant subline was generated from the non-small lung cancer cell line NCI-H460 by stepwise selection in paclitaxel from 0.032 to 250 nmol/L. The resulting subline, designated NCI-H460/PTX250, showed 792-fold resistance against paclitaxel compared with the parental cell line NCI-H460. The chemosensitivity analysis revealed the cross-resistance phenotype against various anticancer drugs including docetaxel, vinblastine, and doxorubicin, but not against camptotecin, cisplatin, and 5-fluorouracil. The addition of 5 mumol/L verapamil or reversin 121 reversed the resistance against paclitaxel, vinblastine, and doxorubicin. The gene expression profile, examined using oligonucleotide microarrays, demonstrated that the expression of 332 and 342 genes was significantly increased and decreased, respectively, in NCI-H460/PTX250 compared with NCI-H460. The most highly upregulated gene was MDR1/ABCB1 with a 1,092-fold increase. The overexpression was confirmed at the protein level by Western blot and flow cytometry analyses. The copy number profile, examined using microarray-based comparative genomic hybridization, revealed amplification of the q11.21 approximately q21.12 region on chromosome 7. In particular, the entire q21.12 region displayed 11- to 13-fold higher copy number in NCI-H460/PTX250 than in NCI-H460. Most of the genes within the region were highly expressed, and the increased expression of these genes could be explained by the amplification in the gene copy number. However, the increase in MDR1/ABCB1 expression greatly exceeded the genomic copy number increase of the gene, suggesting the existence of one or more additional factors, such as transcriptional enhancement or mRNA stabilization, associated with the elevated MDR1/ABCB1 expression. In conclusion, both chromosomal region-specific copy number amplification and gene-specific activation are probably involved in the overexpression of MDR1/ABCB1, resulting in acquisition of the drug resistance phenotype in NCI-H460/PTX250.

Expression of the steroid and xenobiotic receptor and its possible target gene, organic anion transporting polypeptide-A, in human breast carcinoma.

Steroid and xenobiotic receptor (SXR) or human pregnane X receptor (hPXR) has been shown to play an important role in the regulation of genes related to xenobiotic detoxification, such as cytochrome P450 3A4 and multidrug resistance gene 1. Cytochrome P450 enzymes, conjugation enzymes, and transporters are all considered to be involved in the resistance of breast carcinoma to chemotherapeutic or endocrine agents. However, the expression of SXR/hPXR proteins and that of its target genes and their biological or clinical significance have not been examined in human breast carcinomas. Therefore, we first examined SXR/hPXR expression in 60 breast carcinomas using immunohistochemistry and quantitative reverse transcription-PCR. We then searched for possible SXR/hPXR target genes using microarray analysis of carcinoma cells captured by laser microscissors. SXR/hPXR was detected in carcinoma tissues but not in nonneoplastic and stromal cells of breast tumors. A significant positive correlation was detected between the SXR/hPXR labeling index and both the histologic grade and the lymph node status of the carcinoma cases. Furthermore, in estrogen receptor-positive cases, SXR/hPXR expression was also positively correlated with expression of the cell proliferation marker, Ki-67. Microarray analysis showed that organic anion transporting polypeptide-A (OATP-A) was most closely correlated with SXR/hPXR gene expression, and both OATP-A mRNA and protein were significantly associated with SXR/hPXR in both breast carcinoma tissues and its cell lines. These results suggest that SXR/hPXR and its target gene, such as OATP-A, may play important roles in the biology of human breast cancers.

Immunolocalization of liver receptor homologue-1 (LRH-1) in human breast carcinoma: possible regulator of insitu steroidogenesis.

Liver receptor homologue-1 (LRH-1) belongs to a class of nuclear orphan receptor. We examined immunolocalization of LRH-1 in 106 breast carcinomas. LRH-1 immunoreactivity was detected in 43% of the invasive ductal carcinoma. It was negatively correlated with clinical stage, histological grade and HER2 status, and positively associated with sex-steroid receptors, steroidogenic acute regulatory protein, P450 side-chain cleavage, and 3beta-hydroxysteroid dehydrogenase. LRH-1 immunoreactivity was also detected in 28% of the ductal carcinoma in situ. These results suggest that LRH-1 is frequently detected in breast carcinoma tissues, and plays important roles including the regulation of in situ steroidogenesis.

Genes commonly upregulated by hypoxia in human breast cancer cells MCF-7 and MDA-MB-231.

Hypoxia is a stress that causes alterations in signal transduction and gene instability. In the cancer microenvironment, hypoxia plays a significant role in forming a tumor phenotype and tumor progression. We aimed to identify the genes upregulated by hypoxia in human breast cancer cell lines, a hormone-dependent MCF-7 and a hormone-independent MDA-MB-231, using microarray analysis. These cells were exposed to two oxygen concentrations such as 21% and 1% in a time-course. Out of 12625 genes, 26 genes were identified as commonly upregulated in both MCF-7 and MDA-MB-231 cells. Some of these genes were already reported as hypoxia-related, but some of those were identified newly. These commonly upregulated genes between hormone-dependent and hormone-independent cells would be a clue to study hypoxia-related events and to explore the novel therapeutic targets in human breast cancer.

Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene.