Elevated TAK1 augments tumor growth and metastatic capacities of ovarian cancer cells through activation of NF-κB signaling.

Transforming growth factor (TGF)-ß-activating kinase 1 (TAK1) is a serine/threonine kinase which is frequently associated with human cancer progression. However, its functional role in tumorigenesis is still controversial. Here, we report that TAK1 enhances the oncogenic capacity of ovarian cancer cells through the activation of NF-κB signaling. We found that TAK1 is frequently upregulated and significantly associated with high-grade and metastatic ovarian cancers. Mechanistic studies showed that Ser412 phosphorylation is required for TAK1 in activating NF-κB signaling and promotes aggressiveness of ovarian cancer cells. Conversely, suppression of TAK1 activity by point mutation at Ser412, RNAi mediated gene knockdown or TAK1 specific inhibitor ((5Z) -7-Oxozeaenol) remarkably impairs tumor growth and metastasis in ovarian cancer in vitro and in vivo. Our study underscores the importance of targeting TAK1 as a promising therapeutic approach to counteract the ovarian cancer progression.

Reduced expression of AMPK-ß1 during tumor progression enhances the oncogenic capacity of advanced ovarian cancer.

AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism. Our previous study revealed that the subunits of the heterotimeric AMPK enzyme are diversely expressed during ovarian cancer progression. However, the impact of the variable expression of these AMPK subunits in ovarian cancer oncogenesis remains obscure. Here, we provide evidence to show that reduced expression of the AMPK-ß1 subunit during tumor progression is associated with the increased oncogenic capacity of advanced ovarian cancer cells. Immunohistochemical analysis revealed that AMPK-ß1 levels were reduced in advanced-stage (P = 0.008), high-grade (P = 0.013) and metastatic ovarian cancers (P = 0.008). Intriguingly, down-regulation of AMPK-ß1 was progressively reduced from tumor stages 1 to 3 of ovarian cancer. Functionally, enforced expression of AMPK-ß1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion. Conversely, depletion of AMPK-ß1 by siRNA enhanced the oncogenic capacities of ovarian cancer cells, suggesting that the loss of AMPK-ß1 favors the aggressiveness of ovarian cancer. Mechanistically, enforced expression of AMPK-ß1 increased AMPK activity, which, in turn, induced cell-cycle arrest via inhibition of AKT/ERK signaling activity as well as impaired cell migration/invasion through the suppression of JNK signaling in ovarian cancer cells. Taken together, these findings suggest that the reduced expression of AMPK-ß1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer.

FOXM1b, which is present at elevated levels in cancer cells, has a greater transforming potential than FOXM1c.

The forkhead box (FOX) M1 transcription factor is required to maintain the proliferation of cancer cells. Two transcriptionally active isoforms of FOXM1, FOXM1b and FOXM1c, have been identified, but their functional differences remain unclear. FOXM1c is distinguished from FOXM1b by an extra exon (exon Va) that contains an ERK1/2 target sequence. Based on a literature search and quantitative PCR analysis, we concluded that FOXM1b is the predominant isoform that is overexpressed in cancers. The further characterization of FOXM1b and FOXM1c revealed two interesting differences. First, FOXM1b exhibited a higher transforming ability than FOXM1c in a soft agar assay. Second, the transactivating activity of FOXM1c, but not that of FOXM1b, was sensitive to activation by RAF/MEK/MAPK signaling. Importantly, the MEK1 activation of FOXM1c was associated with proteolytic processing to generate short forms that might represent constitutively active forms missing the N-terminal inhibitory domain; in contrast, the proteolytic processing of FOXM1b did not require MEK1 activation. Our findings suggest that FOXM1b is functionally more active. These results provide novel insights into the regulation of FOXM1 activity and its role in tumorigenesis.

Melatonin MT1 receptor-induced transcriptional up-regulation of p27(Kip1) in prostate cancer antiproliferation is mediated via inhibition of constitutively active nuclear factor kappa B (NF-κB): potential implications on prostate cancer chemoprevention and therapy.

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27(Kip1) through dual activation of Gα(s)/protein kinase A (PKA) and Gα(q)/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27(Kip1) in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27(Kip1) promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27(Kip1) promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27(Kip1) transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gα(s)) PKA and (Gα(q)) PKC can de-repress the p27(Kip1) promoter leading to transcriptional up-regulation of p27(Kip1). MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy.

Increased expression of PITX2 transcription factor contributes to ovarian cancer progression.

BACKGROUND: Paired-like homeodomain 2 (PITX2) is a bicoid homeodomain transcription factor which plays an essential role in maintaining embryonic left-right asymmetry during vertebrate embryogenesis. However, emerging evidence suggests that the aberrant upregulation of PITX2 may be associated with tumor progression, yet the functional role that PITX2 plays in tumorigenesis remains unknown. PRINCIPAL FINDINGS: Using real-time quantitative RT-PCR (Q-PCR), Western blot and immunohistochemical (IHC) analyses, we demonstrated that PITX2 was frequently overexpressed in ovarian cancer samples and cell lines. Clinicopathological correlation showed that the upregulated PITX2 was significantly associated with high-grade (P = 0.023) and clear cell subtype (P = 0.011) using Q-PCR and high-grade (P<0.001) ovarian cancer by IHC analysis. Functionally, enforced expression of PITX2 could promote ovarian cancer cell proliferation, anchorage-independent growth ability, migration/invasion and tumor growth in xenograft model mice. Moreover, enforced expression of PITX2 elevated the cell cycle regulatory proteins such as Cyclin-D1 and C-myc. Conversely, RNAi mediated knockdown of PITX2 in PITX2-high expressing ovarian cancer cells had the opposite effect. CONCLUSION: Our findings suggest that the increased expression PITX2 is involved in ovarian cancer progression through promoting cell growth and cell migration/invasion. Thus, targeting PITX2 may serve as a potential therapeutic modality in the management of high-grade ovarian tumor.

Aberrant activation of ERK/FOXM1 signaling cascade triggers the cell migration/invasion in ovarian cancer cells.

Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor essential for cell cycle progression. Numerous studies have documented that FOXM1 has multiple functions in tumorigenesis and its elevated levels are frequently associated with cancer progression. Here, we characterized the role of ERK/FOXM1 signaling in mediating the metastatic potential of ovarian cancer cells. Immunohistochemical (IHC), immunoblotting and semi-quantitative RT-PCR analyses found that both phospho-ERK and FOXM1 were frequently upregulated in ovarian cancers. Intriguingly, the overexpressed phospho-ERK (p<0.001) and FOXM1 (p<0.001) were significantly correlated to high-grade ovarian tumors with aggressive behavior such as metastasized lymph node (5 out of 6). Moreover, the expressions of phospho-ERK and FOXM1 had significantly positive correlation (p<0.001). Functionally, ectopic expression of FOXM1B remarkably enhanced cell migration/invasion, while FOXM1C not only increased cell proliferation but also promoted cell migration/invasion. Conversely, inhibition of FOXM1 expression by either thiostrepton or U0126 could significantly impair FOXM1 mediated oncogenic capacities. However, the down-regulation of FOXM1 by either thiostrepton or U0126 required the presence of p53 in ovarian cancer cells. Collectively, our data suggest that over-expression of FOXM1 might stem from the constitutively active ERK which confers the metastatic capabilities to ovarian cancer cells. The impairment of metastatic potential of cancer cells by FOXM1 inhibitors underscores its therapeutic value in advanced ovarian tumors.

Zic2 synergistically enhances Hedgehog signalling through nuclear retention of Gli1 in cervical cancer cells.

Aberrant activation of Hedgehog (Hh) signalling has been implicated in the pathogenesis of human cancers. However, the cognate molecular mechanisms contributing to this disregulated pathway are incompletely understood. In this study, we showed that Zic2 was frequently over-expressed and associated with high-grade cervical cancer (p = 0.032), high levels of Gli1 (p < 0.001) and CyclinD1 (p < 0.001) by immunohistochemical and quantitative RT-PCR analyses. Further biochemical studies using luciferase reporter, co-immunoprecipitation, subcellular fractionation and immunofluorescence analyses demonstrated that Zic2 can physically interact with Gli1 and retain it in the nucleus, which in turn increases Gli-mediated transcriptional activity. Gain- and loss-of-function analyses of Zic2 showed that Zic2 could increase Hh signalling activity, cell proliferation and anchorage-independent growth ability in cervical cancer cells. Conversely, deletion of the zinc finger domain at C-terminus of Zic2 significantly abrogated its interaction with Gli1, the retention of Gli1 in the nucleus, effects on Hh signalling activity and oncogenic properties in cervical cancer cells. Our findings suggest that Zic2 is a positive modulator increasing Gli1 transcriptional and oncogenic activity by retaining Gli1 in the nucleus of cervical cancer cells.

Differential functions of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v in ovarian carcinogenesis.

PURPOSE: Aberrant overexpression of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v has been found in numerous human cancers. The goal of this study was to characterize the functions of GRB7 and GRB7v in the ovarian carcinogenesis and to investigate the differential roles of GRB7 and GRB7v in the modulation of signaling pathways. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR, Western blot, and immunohistochemical analyses were used to evaluate the levels of GRB7 and GRB7v. The cellular localization, functions, and signaling pathways regulated by GRB7 and GRB7v were investigated by enforced expression of GRB7 and GRB7v. RESULTS: Quantitative reverse transcription-PCR and Western blot analyses showed that GRB7 and GRB7v were frequently upregulated in ovarian cancer samples. The overexpressed GRB7 (P = 0.009) and GRB7v (P = 0.017) were significantly correlated with high-grade ovarian cancer. Immunohistochemical analysis on ovarian cancer tissue array confirmed that the upregulated GRB7 was significantly correlated with high-grade ovarian cancer (P = 0.001). Confocal microscopy analysis showed that GRB7 and GRB7v predominately localized in cytoplasm of ovarian cancer cells, consistent with their roles as signaling adaptors. Enforced expression of GRB7 promoted cell proliferation, migration, and invasion, whereas GRB7v only increased cell proliferation and anchorage-independent growth ability. With the treatment of specific kinase inhibitors, we showed that both GRB7 and GRB7v promoted cell proliferation through activating extracellular signal-regulated kinase signaling, whereas GRB7 enhanced cell migration/invasion by activating c-Jun NH(2) terminal kinase signaling. CONCLUSIONS: Our studies implicate that the overexpressed GRB7 and GRB7v are associated with high-grade tumors and exert distinct tumorigenic functions through regulating different signaling pathways in ovarian cancer cells.

Age-related decline in stress responses of human myocardium may not be explained by changes in mtDNA.

Elderly patients undergoing cardiac surgery are more likely to suffer postoperative heart failure than younger patients. This phenomenon is mirrored by an age-related loss of mitochondrial function and by an in vitro loss of myocardial contractile force following a stress. To examine the possibility that loss of mtDNA integrity may be responsible, we quantified representative age-associated mtDNA mutations (mtDNA(4977) and mtDNA(A3243G)) and mtDNA copy number using quantitative polymerase chain reaction in atrial samples obtained during cardiac surgery. The myocardium underwent organ bath contractility testing before and after either an ischaemic or hypoxic stress. We found that with age, recovery of developed force after either stressor significantly declined (p<0.0001). The abundance of mtDNA(4977) correlated weakly with loss of contractility (R(2)=0.09, p=0.047). However, the abundance level was low (average 0.0075% of total mtDNA) and the correlation disappeared when age was included in a multivariate analysis. Neither the abundance of mtDNA(A3243G) nor mtDNA copy number correlated with reduced recovery of developed force after stress. We conclude that, although mtDNA mutations (as exemplified by mtDNA(4977)) accumulate in the ageing heart, they are unlikely to make a major contribution to loss of contractile function.

Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis: a differential expression and functional analysis.

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G(1) cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1-induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1-transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis.

Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells.

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.

Melatonin as a negative mitogenic hormonal regulator of human prostate epithelial cell growth: potential mechanisms and clinical significance.

Circannual variation in the human serum levels of prostate-specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT(1) and MT(2) receptors was detected in benign human prostatic epithelial tissues and RWPE-1 cells. MLT and 2-iodomelatonin inhibited RWPE-1 cell proliferation and up-regulated p27(Kip1) gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT(1)/MT(2) receptor antagonist luzindole, but were not affected by the selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE-1 cells was effected via increased p27(Kip1) gene transcription through MT(1) receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT(1)/PKA+PKC/p27(Kip1) antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor-mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth.

Detection of mosaic pattern of mitochondrial DNA alterations in different populations of cells from the same endometrial tumor.

Somatic mitochondrial DNA (mtDNA) alterations including point mutations and microsatellite instability (MSI) have been frequently detected in human cancers. To further explore the extensiveness of mtDNA alterations, we have analyzed the occurrence of somatic mtDNA mutations in different populations of endometrial cancer cells from the same tumor tissues as compared with adjacent non-tumor cells. Laser-captured micro-dissection was used to harvest endometrial cancer cells from separated areas of the same tumor and adjacent normal cells. Total DNA isolated from micro-dissected cells was PCR amplified and analyzed for mtDNA alterations by polyacrylamide gel electrophoresis and DNA sequencing. Multiple mtDNA alterations were detected in different portions of the same tumor. Different populations of endometrial cancer cells carried different patterns of mtDNA mutations. Interestingly, unlike previous reports, most mutations were found to be heteroplasmic. We have demonstrated the occurrence of hyper-variability of mtDNA alterations in a single piece of tumor tissue. Our observations support the hypothesis that the accumulation of mtDNA alterations is random and expands independently. The data presented here showed the heterogeneity of cancer cells in terms of mtDNA alterations in endometrial cancer.

Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data.

BACKGROUND: Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. METHODS: For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers) was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using chi2 or Fisher's exact test. RESULTS: APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2), endometrial cancer (CASP8, CDH13, hMLH1 and p73), and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1). The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers) were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer) to the highest 56.7% (DAPK in cervical cancer). Aberrant methylation for some genes (BRCA1, DAPK, hMLH1, MGMT, p14, p16, and PTEN) was also associated with clinico-pathological data. CONCLUSION: Thus, differential methylation profiles occur in the three types of gynecologic cancer. Detection of methylation for critical loci is potentially useful as epigenetic markers in tumor classification. More studies using a much larger sample size are needed to define the potential role of DNA methylation as marker for cancer management.

Semi-quantitative fluorescent PCR analysis identifies PRKAA1 on chromosome 5 as a potential candidate cancer gene of cervical cancer.

OBJECTIVE: Comparative genomic hybridization has frequently detected amplification of chromosome 5p in cervical cancer, but candidate cancer genes within the region are rarely known. Therefore, we pursued to identify potential candidate gene related to cervical cancer development. METHODS: A series of 128 cervical tumor samples were examined by semi-quantitative fluorescent differential PCR for copy number changes on three candidate genes (PRKAA1, CTNND2 and POLS) mapped to chromosome 5p and one gene (ERBIN) mapped to chromosome 5q12.3. The impact of gene copy number was later analyzed in relation to HPV infection, tumor stage or tumor radiosensitivity. RESULTS: DNA copy numbers of PRKAA1, CTNND2 and ERBIN were significantly different from normal controls (P < 0.05). DNA copy number changes did not correlate with HPV infection, tumor stages or tumor radiosensitivity. Using RT-PCR, PRKAA1 mRNA expression in seven tumor samples with known 5p amplification was amplified from 3- to 15-fold. Over-expression of PRKAA1 was further confirmed by immunohistochemical staining on 125 paraffin-embedded cervical cancer tissues. The expression level in cervical tumor was significantly higher than that in normal epithelium (P < 0.001). CONCLUSIONS: PRKAA1 gene codes for the catalytic alpha 1 subunit of the AMP-activated protein kinase which is an important cellular metabolic stress regulator. It might assist tumor cells growth under stress. Thus, PRKAA1 may be one of the potential candidate genes for cervical carcinogenesis.

Mitochondrial DNA deletion in granulosa and cumulus oophorus cells.

The incidence of the 4,977-bp deletion in mitochondrial DNA (DeltamtDNA4977) in 73 pairs of granulosa and cumulus oophorus cells was studied with polymerase chain reaction (PCR), and was significantly higher in granulosa cells (GC) (67/73, 91%) than in cumulus oophorus cells (17/73, 23.3%), independent of the donors' age. In the cumulus oophorus cells, the oocyte morphology, the ooplasma diameter, and the proportion of oocytes fertilized normally were comparable between those with and without DeltamtDNA4977; whereas the oocyte diameter and the zona thickness were significantly higher in those with DeltamtDNA4977.

Frequent occurrence of mitochondrial microsatellite instability in the D-loop region of human cancers.

We analyzed the occurrence of mitochondrial microsatellite instability (mtMSI) in 262 pairs of female cancer tissues with the matched normal controls. mtMSI was detected in only 4 of 12 microsatellites found in the mitochondrial genome (3 in the D-loop and 1 in the 12S rRNA gene). Interestingly, 95.6% (87/91) of mtMSI was detected in the D-loop, namely, at nucleotide positions 303-315, 514-523, and 16184-16193. This demonstrates that the D-loop is a hotspot for mtMSI. Different incidences of mtMSI at these three microsatellites were found in the four cancer types (including cervical, endometrial, ovarian, and breast). Together with those mtMSI reported in other studies, the differential occurrence of mtMSI at each of the markers in the D-loop region was observed, indicating that the extent of mtMSI varies from one cancer to another. Although the mechanisms of generation and functional impact of mtMSI are still not clear, the high incidence of mtMSI in the D-loop and its broad distribution in human cancers render it a potential marker for cancer detection.

The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: a quantitative study using laser-captured microdissected tissues.

OBJECTIVE: Microsatellite instability (MSI) is a frequent genetic event in the D-loop region (which controls mitochondrial DNA (mtDNA) replication) of mitochondrial genome of endometrial cancer. We therefore investigated the relationship between mtMSI and mtDNA content in endometrial cancer. METHODS: Tumor tissues from 65 cancer patients and normal tissues from 41 non-cancer patients were used in this study. Pure endometrial adenocarcinoma cells and normal endometrial glandular epithelial cells were collected by laser capture microdissection, and analyzed for levels of mtDNA copy number by real-time quantitative PCR. RESULTS: Our data show that mtDNA copy number was not related with age in both endometrial cancer and normal endometrium cells. Great inter-individual variations in mtDNA copy number in endometrial cancer group were found; and mtDNA content was significantly larger than that in normal endometrium group. About 2-fold increase of mtDNA copy number was found in endometrial adenocarcinoma compared with normal endometrial glandular epithelium (P = 0.001). In particular, the analysis also shows that the copy number of mtDNA in the cases that carried the mtMSI at nucleotide position 303 was significantly higher than that of the negative cases (P = 0.048). CONCLUSIONS: Our data indicate that mtDNA copy number increased during endometrial cancer development. There is also a correlation between the mtDNA instability and mtDNA content in endometrial cancer cells. Role of mitochondrial genome changes in carcinogenesis warrants further investigation.

Detection of mitochondrial DNA mutations in gestational trophoblastic disease.

Mitochondrial DNA (mtDNA) mutations have been implicated in a wide range of human disease. However, its role in gestational trophoblastic disease remains unclear. In this study, the entire mitochondrial genome of 10 hydatidiform moles (HM) and one choriocarcinoma were examined by automated DNA sequencing after amplification by polymerase chain reaction. MtDNA sequences obtained separately from disease tissues (HM and choriocarcinoma) and patients' tissues were compared. Of the 133 neutral sequence variants identified, 41 have not been reported to date. Large or small-scale deletion or insertion was not detected in any of the samples studied. A total of six (five in the D-loop and one in the 16S rRNA gene) somatic point mutations were detected in the choriocarcinoma sample, in contrast to none being detected in the HM samples. Somatic mtDNA instability was detected in the D-loop region in three cases of HM as well as in the choriocarcinoma sample. Somatic mtDNA instability appeared in the same nucleotide position, from 303 to 309, within the Conserved Sequence Block II resulting in alteration in length of the homopolymorphic C-tract, reflecting microsatellite instability. The results suggest that mtDNA instability may be an early event occurring at a premalignant stage. Occurrence of multiple somatic mtDNA mutations in choriocarcinoma suggests that mtDNA mutations might play an important role in the molecular pathogenesis of invasive gestational trophoblastic disease.