Lih-Ching Hsu

Loss of distal 11q is associated with DNA repair deficiency and reduced sensitivity to ionizing radiation in head and neck squamous cell carcinoma

About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage‐fusion‐bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of γ‐H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor.

Centrosome abnormalities in ovarian cancer

Centrosome abnormalities have been found in various cancer types and are thought to be involved in early development of cancer and/or progression. The contribution of centrosome abnormalities to ovarian tumorigenesis has not been previously evaluated. We sought to determine whether centrosome dysfunction occurs in ovarian tumorigenesis, and whether it could be used as an indicator of early neoplastic changes in ovarian surface epithelium (OSE). Primary cultures of normal OSE and ovarian tumors, as well as paraffin‐embedded normal ovaries and ovarian tumors of different stages, were used for immunostaining with a γ‐tubulin antibody. Centrosomes were considered abnormal if there were more than 2 per cell, if their sizes were greater than 2‐fold of a normal centrosome, and/or if they were abnormal in shape. Centrosomes in normal tissue were uniform in size, whereas centrosomes in ovarian tumors tended to be abnormal in size, number and shape. On average, 4.7% of cells in 5 primary normal OSE cultures had more than 2 centrosomes, whereas 14.1% of primary cells from 5 ovarian tumors displayed centrosome abnormalities (p = 0.008). Centrosome abnormalities were present in 60.9% of stage I (n = 23), 83.3% of stage II (n = 30) and all stage III (n = 10) paraffin‐embedded ovarian tumor samples examined, but not in normal tissues. In addition, centrosome abnormalities occurred more frequently in ovarian tumors with higher grade and aggressive serous subtype. This is the first demonstration that centrosome abnormalities occur in ovarian tumors. Centrosome dysfunction may be an early event in ovarian carcinogenesis and involved in ovarian tumor progression.

Gene amplification and overexpression of protein phosphatase 1α in oral squamous cell carcinoma cell lines

Gene amplification of chromosomal band 11q13 is observed frequently in oral squamous cell carcinomas (OSCC). Several genes have been identified in the 11q13 amplicon, including FGF3, FGF4, CCND1, EMS1 and TAOS1. Some of these genes show good correlation between gene copy number and gene expression, and are thought to play a role in driving 11q13 amplification. The PPP1CA gene, which encodes the catalytic subunit of serine/threonine protein phosphatase protein phosphatase 1α (PP1α), is also located in 11q13. Protein phosphatase 1α, one of the isoforms of PP1, regulates critical cellular events, such as cell cycle progression, and apoptosis. We sought to explore the possibility that PPP1CA was amplified and overexpressed in OSCC cells. Indeed, some OSCC cell lines had PPP1CA gene amplification, as analysed by fluorescence in situ hybridization. We have also demonstrated that PPP1CA gene copy number is increased in 21% of the OSCC cell lines determined by quantitative microsatellite analysis. PP1α RNA expression determined by quantitative reverse transcription–polymerase chain reaction was significantly higher in OSCC cell lines with 11q13 amplification compared to those without 11q13 amplification (P=0.011). The difference was even more significant between cell lines with at least three copies of the PPP1CA gene and those with less than three copies of the gene (P=0.00045). Relative PP1α protein levels were also significantly associated with PPP1CA gene copy number (P=0.014). Furthermore, knockdown of PP1α and/or cyclin D1 by small interfering RNA suppressed OSCC cell growth, at least in part by modulating pRB phosphorylation, resulting in G0 growth arrest. These data suggest that like the cyclin D1 gene, CCND1, amplification and overexpression of the PP1α gene, PPP1CA, may be involved in OSCC tumorigenesis and/or progression.

Allelic imbalance in mammary carcinomas induced by either 7,12‐dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis

To identify and compare the genetic lesions associated with tumorigenesis in rats carrying the mammary carcinoma suppressor (MCS) 1 gene, we induced mammary carcinomas in (Wistar Furth (WF) × Copenhagen (Cop))F1 rats by using either 7,12‐dimethylbenz[a]anthracene (DMBA) or radiation. The tumors were screened for allelic imbalances by using polymerase chain reaction and 65 polymorphic microsatellite markers spanning the genome. No allelic imbalance was detected at the mapped location of MCS‐1 on chromosome 2; however, a scan of the genome revealed random allelic imbalances in the radiation‐induced tumors. In addition, non‐random loss of heterozygosity (LOH) on chromosome 1 in the DMBA‐induced tumors was documented. We then screened three other subsets of DMBA‐ and radiation‐induced mammary carcinomas from (WF × Fischer (F344))F1, (Wistar Kyoto×F344)F1, and (F344×Cop)F1 rats for imbalance on chromosomes 1 and 2. No allelic imbalance was detected in the MCS‐1 region of chromosome 2 in any of the tumors screened. Nonrandom imbalance on chromosome 1 was detected but only in the DMBA‐induced tumors from the (F344×Cop)F1 rats. Thus, only Cop‐derived F1 rats have mammary tumors with the chromosome 1 imbalance; however, the imbalance does not favor the Cop parental allele. We also analyzed the DMBA‐induced tumors with LOH at chromosome 1 for Ha‐ras codon 61 mutation and found no association. These results suggest that loss of the MCS‐1 Cop allele is not required for tumor formation, that the genetic background of the F1 rat appears to influence the type of genetic lesion identified in the mammary tumors, and that there is no association between Ha‐ras codon 61 mutation and chromosome 1 imbalance in our model system.

Coordinate up-regulation of TMEM97 and cholesterol biosynthesis genes in normal ovarian surface epithelial cells treated with progesterone: implications for pathogenesis of ovarian cancer

BackgroundOvarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood.MethodsTo determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts.ResultsWe identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells.ConclusionThese findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries

Abstract. Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat.

Pim-1 knockdown potentiates paclitaxel-induced apoptosis in human hormone-refractory prostate cancers through inhibition of NHEJ DNA repair.

The knockdown of Pim-1 or inhibition of Pim-1 activity significantly increased γ-H2A.X expression. The effect was correlated to apoptosis and was attributed to the inhibition of nonhomologous DNA-end-joining (NHEJ) repair activity supported by the following observations: (1) inhibition of ATM and DNA-PKcs activities, (2) down-regulation of Ku expression and nuclear localization and (3) decrease of DNA end-binding of both Ku70 and Ku80. The data suggest that Pim-1 plays a crucial role in the regulation of NHEJ repair. In the absence of Pim-1, the ability of DNA repair significantly decreases when exposed to paclitaxel, leading to severe DNA damage and apoptosis.

Reevesioside A, a cardenolide glycoside, induces anticancer activity against human hormone-refractory prostate cancers through suppression of c-myc expression and induction of G1 arrest of the cell cycle.

In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of Reevesia formosana, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r2 = 0.98) between the expression of Na+/K+-ATPase α3 subunit and anti-proliferative activity suggested the critical role of the α3 subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells.

Ardisianone, a natural benzoquinone, efficiently induces apoptosis in human hormone-refractory prostate cancers through mitochondrial damage stress and survivin downregulation

Increasing evidence suggests that mitochondria play a central role in regulating cell apoptosis. Survivin, an inhibitor of apoptosis protein (IAP) family member, mediates resistance to cancer chemotherapy particularly in prostate cancers. Therefore, development of anticancer agents targeting mitochondria and survivin is a potential strategy.

Reevesioside F induces potent and efficient anti-proliferative and apoptotic activities through Na+/K+-ATPase α3 subunit-involved mitochondrial stress and amplification of caspase cascades

Reevesioside F, isolated from Reevesia formosana, induced anti-proliferative activity that was highly correlated with the expression of Na⁺/K⁺-ATPase α₃ subunit in several cell lines, including human leukemia HL-60 and Jurkat cells, and some other cell lines. Knockdown of α₃ subunit significantly inhibited cell apoptosis suggesting a crucial role of the α₃ subunit. Reevesioside F induced a rapid down-regulation of survivin protein, followed by release of cytochrome c from mitochondria and loss of mitochondrial membrane potential (ΔΨm). Further examination demonstrated the mitochondrial damage in leukemic cells through Mcl-1 down-regulation, Noxa up-regulation and an increase of the formation of truncated Bid, tBim and a 23-kDa cleaved Bcl-2 fragment. Furthermore, reevesioside F induced an increase of mitochondria-associated acetyl α-tubulin that may also contribute to apoptosis. The caspase cascade was profoundly activated by reevesioside F. Notably, the specific caspase-3 inhibitor z-DEVD-fmk significantly blunted reevesioside F-induced loss of ΔΨm and apoptosis, suggesting that caspase-3 activation may further amplify mitochondrial damage and apoptotic signaling cascade. In spite of being a cardiac glycoside, reevesioside F did not increase the intracellular Ca²⁺ levels. Moreover, CGP-37157 which blocked Na⁺/Ca²⁺ exchanger on plasma membrane and mitochondria did not modify reevesioside F-mediated effect. In summary, the data suggest that reevesioside F induces apoptosis through the down-regulation of survivin and Mcl-1, and the formation of pro-apoptotic fragments from Bcl-2 family members. The loss of ΔΨm and mitochondrial damage are responsible for the activation of caspases. Moreover, the amplification of caspase-3-mediated signaling pathway contributes largely to the execution of apoptosis in leukemic cells.