Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer.

BACKGROUND: Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. METHODS: FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. RESULTS: Quantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21(WAF1/CIP1) in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-beta-induced p21(WAF1/CIP1) expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21(WAF1/CIP1) expression. Notably, FOXG1 was able to inhibit the p21(WAF1/CIP1) promoter activity in a p53-independent manner by transient reporter assays. CONCLUSION: Our results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-beta-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21(WAF1/CIP1) transcription.

Methylation profile in benign, borderline and malignant ovarian tumors.

PURPOSE: Promoter hypermethylation is a common phenomenon in neoplasm. The aims of this study were (a) to compare the methylation profiles in different types of ovarian tumors and (b) to determine the possible relationship between the methylation status and different clinicopathologic characteristics. METHODS: We examined the promoter methylation status of 9 tumor suppressor genes (RARbeta2, TMS1, RIZ1, P15, P16, PTEN, MINT31, APC and HIC1) in 89 ovarian cancers, 16 borderline ovarian tumors, 19 benign ovarian tumors, 16 normal ovarian tissue and 5 ovarian cancer cell lines. The methylation status was examined with respect to clinicopathologic characteristics of the ovarian cancer patients. RESULTS: Methylation indices for ovarian cancer, borderline ovarian tumor, benign ovarian tumor, normal ovarian tissue and ovarian cancer cell lines were 28.8, 20.1, 10.5, 11.8 and 42.2%, respectively. It was significantly higher in ovarian cancer, borderline ovarian tumor and ovarian cancer cell lines (X (2) test, P < 0.001, P = 0.01 and P < 0.001, respectively) than benign or normal ovarian tissues. In ovarian cancer, concurrent methylation of at least two genes (CM2) was associated with early stage disease (X (2) test, P = 0.035) and less recurrence (X (2) test, P = 0.020). When the methylation statuses of the nine genes as well as CM2 were included in multivariate Cox Regression analysis, CM2 was the only independent predictor for survival (P = 0.013). CONCLUSION: CM2 was an independent predictor for survival in ovarian cancer.

High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas.

To investigate the potential role of somatic mitochondrial DNA (mtDNA) mutations in tumorigenesis, the occurrence of mutations in mtDNA of ovarian carcinomas was studied. We sequenced the D-loop region of mtDNA of 15 primary ovarian carcinomas and their matched normal controls. Somatic mtDNA mutations were detected in 20% (3 of 15) tumor samples carrying single or multiple changes. Complete sequence analysis of the mtDNA genomes of another 10 pairs of primary ovarian carcinomas and control tissues revealed somatic mtDNA mutations in 60% (6 of 10) of tumor samples. Most of these mutations were homoplasmic, and most were T-->C or G-->A transitions, but one represented a differential length within a run of identical C residues. A region of mtDNA sequence including the 16S and 12S rRNA genes, the D-loop and the cytochrome b gene, may represent the zone of preferred mtDNA mutation in ovarian cancer. The high incidence of mtDNA mutations found in ovarian carcinomas and other human cancers suggests that genetic instability of mtDNA might play a significant role in tumorigenesis.

Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis.

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over-expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over-expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1-deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage-independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1-over-expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage-independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27(Kip1) and p21(Cip1). Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC.

Association of decreased mitochondrial DNA content with ovarian cancer progression.

Mitochondrial DNA (mtDNA) content in ovarian carcinomas was assessed by quantitative PCR. Results show that mtDNA content in tumour cell was significantly higher than that in normal ovary. Change in mtDNA content was not related with patients' age or tumour stages. However, the average mtDNA copy number in pathological low-grade tumours was over two-fold higher than that in high-grade carcinomas (P=0.012). Moreover, type I carcinomas also had a significantly higher mtDNA copy number than in type II carcinomas (P=0.019). Change in mtDNA content might be an important genetic event in the progression of ovarian carcinomas.

Microsatellite instability in mitochondrial genome of common female cancers.

To investigate the occurrence of mitochondrial genome instability in primary cervical, endometrial, ovarian, and breast carcinomas, we analyzed 12 microsatellite regions in mitochondrial DNA (mtDNA) of tumor tissues and their matched normal controls. Four of the 12 microsatellite markers starting at nucleotide position (np) 303, 514, 956, and 16184, respectively, exhibited instability as indicated by the change in length of short base-repetitive sequences of mtDNA in cancer tissue relative to that in control normal tissue from the same patient. About 25.4% of cervical cancers, 48.4% of endometrial cancers, 21.9% of ovarian cancers, and 29.4% of breast cancers carried one or more mitochondrial microsatellite instability (mtMSI). mtMSI was frequently detected in the D-loop region but rarely occurred in the coding region. A relatively long C tract interrupted by a T residue is the mtMSI hot spot in all four types of cancer studied. Different tumors have different mtMSI profiles. In particular, the frequency of mtMSI in endometrial cancer was significantly higher than in the other three types of cancer. Furthermore, carriers of a germ-line T to C polymorphism at np 16189 could be more susceptible to breast cancer development in light of the higher frequency detected in cancer patients than in normal individuals.

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes.

Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Gross mosaic pattern of mitochondrial DNA deletions in skeletal muscle tissues of an individual adult human subject.

Using polymerase chain reaction techniques, the patterns of mitochondrial DNA deletions (which characteristically occur at low abundance during ageing) were compared in different skeletal muscle samples of an adult human subject. In one particular section of the biceps muscle, an unusual pattern of mitochondrial DNA deletions was detected; the common 4977 bp deletion was absent, but other deletions were observed. In pectoralis (chest) muscle, deletions commonly seen in normal adults were readily detected, including the 4977 bp deletion. Significantly, different patterns of mtDNA deletions were found in two adjacent parts of the same biceps muscle sample: one was the unusual pattern mentioned above, but the other part clearly contained the 4977 bp deletion. The results therefore demonstrate a gross mosaic pattern of mtDNA deletions in the skeletal muscle tissues of an individual human subject.

Risk of cervical cancer is not increased in Chinese carrying homozygous arginine at codon 72 of p53.

Homozygous arginine at codon 72 (HA72) of p53 was found in 22% of normal cervices and 30.0% of cervical cancers and no significant difference was detected between normal and cervical cancer with or without HPV 16/18. There was no correlation between HA72 and risk of cervical cancer in Chinese.

Mutations in mitochondrial DNA accumulate differentially in three different human tissues during ageing.

In 60 human tissue samples (encompassing skeletal muscle, heart and kidney) obtained from subjects aged from under 1 to 90 years, we used quantitative PCR procedures to quantify mitochondrial DNA (mtDNA) molecules carrying the 4977 bp deletion (mtDNA4977) and 3243 A-->G base substitution. In addition, the prevalence of multiple mtDNA deletions was assessed in a semi-quantitative manner. For all three tissues, the correlations between the accumulation of the particular mtDNA mutations and age of the subject are highly significant. However, differential extents of accumulation of the two specific mutations in the various tissues were observed. Thus, the mean abundance (percentage of mutant mtDNA out of total mtDNA) of mtDNA4977in a subset of age-matched adults is substantially higher in skeletal muscle than in heart and kidney. However, the mean abundance of the 3243 A-->G mutation in skeletal muscle was found to be lower than that in heart and kidney. Visualisation of arrays of PCR products arising from multiple mtDNA deletions in DNA extracted from adult skeletal muscle, was readily made after 30 cycles of PCR. By contrast, in DNA extracted from adult heart or kidney, amplification for 35 cycles of PCR was required to detect multiple mtDNA deletions. Although such multiple deletions are less abundant in heart and kidney than in skeletal muscle, in all tissue extracts there are unique patterns of bands, even from different tissues of the same subject. The differential accumulation of mtDNA4977, other mtDNA deletions and the 3243 A-->G mutation in the three tissues analysed presumably reflects different metabolic and senescence characteristics of these various tissues.

Restriction fragment length polymorphisms associated with factor VIII:C gene in Chinese.

Twenty-six unrelated hemophilia A and 70 unrelated normal chromosomes in 184 subjects were studied to determine the frequencies of intragenic and intergenic restriction fragment length polymorphisms associated with the factor VIII:C gene. The incidences for positive BclI and BglI polymorphic sites in the Chinese were 82% and 100%, respectively. Both were higher than in other ethnic groups, while the incidence for XbaI polymorphism was 57%, which is similar to that reported in Caucasians. Using the St14.1 probe, two polymorphic TaqI allelic systems in the DXS52 region were detectable, with heterozygous rates of 0.712 (for system I, alleles 1 to 8) and 0.495 (for system II, alpha and beta alleles), respectively. Thus, using a combination of four polymorphisms, it would be possible to offer carrier detection or prenatal diagnosis in 96% of Chinese females at risk.

Quantitative allele-specific PCR: demonstration of age-associated accumulation in human tissues of the A-->G mutation at nucleotide 3243 in mitochondrial DNA.

We have developed an improved allele-specific polymerase chain reaction (AS-PCR) procedure that can selectively amplify mutant DNA sequences (which differ from the normal sequences by a single base pair) in the presence of large excess of normal sequences. We applied this procedure to quantification of mutant molecules of human mitochondrial DNA (mtDNA). Conditions for AS-PCR have been systematically varied, encompassing DNA template input, annealing temperature, and PCR cycle number. Adjustment of these three reaction parameters to optimal conditions, using plasmids containing cloned segments of mutant and normal mtDNA, enabled the reliable detection of as little as 0.01% of mutant mtDNA. By standardising the DNA input for AS-PCR, the percentage of mutant molecules can be accurately quantified. This improved procedure was used here to detect and quantify the base substitution at nucleotide position 3243 (A-->G) in mtDNA from total cellular DNA isolated from various tissues of both infants and adults. We observed a 5- to 10-fold higher mutant mtDNA (3243 A-->G) frequency in adult tissues than in infant tissues. The results are consistent with the hypothesis that the accumulation of mtDNA mutations is an important feature of the human aging process. The quantitative and sensitive allele-specific amplification system described here is applicable to the quantification of low levels of somatic mutations in oncogenes and tumour suppressor genes in the context of human mutation, and could be extended to any biological situation in which only a small proportion of a DNA molecular population is subjected to a particular base substitution.

Mitochondrial DNA deletions in human cardiac tissue show a gross mosaic distribution.

The variability of mitochondrial DNA (mtDNA) deletional patterns has been investigated in adjacent slices of human heart atrium. Using quantitative PCR we found differential abundances of one particular mtDNA deletion, that of 4977 bp (mtDNA4977), among sets of adjacent slices of right atrial trabeculae pectinatae from 10 subjects. Some subjects had relatively constant abundance of mtDNA4977 among the tissue slices, while others covered a wide range. A qualitative PCR procedure was used to visualize the patterns of multiple deletions within an 8.64-kb segment of the mtDNA genome, in the same set of atrial trabeculae samples. Some subjects showed completely different multiple deletional patterns in each of the trabeculae slices analyzed. There was no correlation between the variation of the abundance of mtDNA4977 and that of the multiple deletions. The results are consistent with the notion that the occurrence of mtDNA deletions during aging is a random process, involving their production throughout the lifetime of an individual. In this view, the patterns of new deletions are superimposed on those already accumulated by propagation and segregation of mutations formed earlier in life.

Long-term follow-up of chronic hepatitis B patients treated with interferon alfa.

BACKGROUND: There is very little data on the long-term outcome of patients with chronic hepatitis B after interferon therapy. A 6-year follow-up of two interferon trials in chronic hepatitis B patients is reported. METHODS: One hundred twenty-eight Chinese adults with chronic hepatitis B who received interferon therapy were followed for 19-79 months (median 41 months). Twenty-nine patients lost hepatitis B e antigen and two also lost hepatitis B surface antigen within 1 year of treatment. RESULTS: Seven (24%) responders reactivated. Twenty-eight (28%) nonresponders had sustained clearance of hepatitis B e antigen during follow-up. Delayed clearance of hepatitis B e antigen occurred more frequently in nonresponders who had elevated pretreatment serum transaminase levels. (P = 0.002). Serum hepatitis B virus DNA became undetectable by polymerase chain reaction assay in both responders who lost hepatitis B surface antigen but in only 8 (17%) patients who lost hepatitis B e antigen only. Delayed clearance of hepatitis B surface antigen was not seen in any of the 48 patients who had sustained clearance of hepatitis B e antigen. CONCLUSIONS: Contrary to reports from Western countries, complete elimination of markers of hepatitis B virus infection was uncommon in Chinese patients with chronic hepatitis B who underwent interferon therapy despite similar duration of follow-up.

Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging.

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease-associated point mutations studied, 3243 A-->G showed significant accumulation with age (P < 0.05), while 8993 T-->G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease-associated) showed no age-associated accumulation: two (7029 C-->T and 7920 A-->G) showed little change over the years (P > 0.1), while the other (13167 A-->G) showed a significant decrease (P < 0.05). both the 4,977-bp and 7,436-bp deletions showed a significant age-associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20-40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age-associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging.

Homozygous arginine at codon 72 of p53 has no prognostic significance in cervical cancer.

Mutation of p53, a tumour suppressor gene, is uncommon in cervical cancer but the detection of human papillomavirus (HPV) DNA in cervical cancer is common. The findings of increased susceptibility to degradation of p53 by E6 protein of HPV16/18 in cervical cancer with homozygous arginine at codon 72 (HA72) of p53 led to this study on whether cervical cancers with HA72 were more aggressive with the increase in the rate of loss of p53 function. In 102 cervical cancers, 76.5% were HPV16/18 positive and 30% had HA72. No survival difference was detected between HA72 and non-HA72 tumours irrespective of HPV16/18 status. Furthermore, the detection of HPV16/18 in cervical cancer was found not to be of prognostic significance in this study.

Low incidence of HPV DNA in sera of pretreatment cervical cancer patients.

OBJECTIVE: The aim of this study was to investigate the feasibility of using DNA in the circulation as a diagnostic tool for cervical cancer. METHODS: We used PCR followed by Southern hybridization to detect human papillomavirus (HPV) DNA in serum samples taken from patients of cervical cancer before treatment. RESULTS: A total of 60 samples were analyzed. In a set of 40 samples, without knowledge of the HPV DNA status in the corresponding cervical carcinomas, we could detect 8 (20%) positive samples, of which 7 (17.5%) were HPV 16 and 1 (2.5%) was HPV 18. In another set of 20 samples, known to be HPV 16 infected in the corresponding cervical carcinomas, we detected only 4 (20%) HPV-16-positive samples. The occurrence of HPV DNA in sera of cervical cancer patients seems sporadic. CONCLUSION: The low incidence indicates that serum HPV DNA has limited application in the diagnosis of cervical cancer.

Primary peritoneal malignant mixed Müllerian tumors. A clinicopathologic, immunohistochemical, and genetic study.

BACKGROUND: Primary peritoneal malignant mixed Müllerian tumors (MMMTs) are rarely reported in the literature. METHODS: The clinical, pathologic, and immunohistochemical features of five cases of MMMT of female peritoneum were analyzed. The tumors were also investigated for expression of hormone receptors, specific BRCA-1 mutations, and clonality. RESULTS: The patients' ages ranged from 33 to 67 years. They presented with abdominal pain or mass. One case of peritoneal MMMT was associated with a synchronous endometrial carcinoma whereas another case was detected 2 years after the diagnosis of a primary adenocarcinoma of the fallopian tube. One patient died 1 month after diagnosis whereas 2 patients died with disease within 1 year. Both carcinomatous and sarcomatous elements are present in all the tumors. Squamous differentiation was noted in two cases. Heterologous elements, including chondroid, rhabodomyoblastic, and osteoid differentiation were detected in all tumors. Immunohistochemical studies confirm the biphasic differentiation with variable demonstration of neural and smooth muscle differentiation. All five MMMTs were negative for estrogen and progestogen receptors although the related endometrial and tubal carcinomas were positive. Heteroduplex analysis used to screen for specific BRCA-1 mutations were negative in all five MMMTs. Clonality study of the two MMMTs found in association with endometrial carcinoma and tubal carcinoma was inconclusive. CONCLUSIONS: Our study confirmed that primary peritoneal MMMTs were aggressive tumors with poor prognosis. The presence of synchronous or metachronous genital carcinomas suggests multifocal tumorigenesis from tissue of same embryologic origin. The lack of hormone receptor in these tumors indicates deviation from hormonal control. Specific BRCA-1 mutations found in ovarian carcinoma in Chinese patients could not be detected in our series.

Molecular analysis of the androgen receptor gene in Hong Kong Chinese infertile men.

PURPOSE: To investigate the relationship between CAG repeat length in the androgen receptor gene and impaired spermatogenesis in Hong Kong Chinese population. METHODS: The CAG repeat region was amplified by polymerase chain reaction (PCR) in 85 nonobstructive azoospermic or severe oligozoospermic men, and 45 fertile males. The number of CAG repeat was analyzed by DNA sequencing. Serum FSH, LH, and testosterone levels were also determined in these men. RESULTS: Among nonobstructive azoospermic males, three men (5.7%) possessed short CAG repeats (< 16), and three (5.7%) other men possessed long CAG repeats (> 30). Short CAG repeats (< 16) were also found in two severe oligozoospermic males (6.3%). The incidence of infertile men with short or long CAG repeats is significantly higher in the azoospermic group (p = 0.03) but not in the severe oligozoospermic group (p = 0.17) when compared with the fertile controls CONCLUSION: Our data suggest an association between CAG repeat lengths and impaired spermatogenesis in azoospermic males in our population.

Independent occurrence of somatic mutations in mitochondrial DNA of human skin from subjects of various ages.

The incidence (frequency of occurrence) and abundance (percentage of mutant out of total mtDNA population) of two different somatic mtDNA mutations in human skin were investigated in 44 subjects ranging from 19 to 87 years of age. Using quantitative allele-specific polymerase chain reaction (AS-PCR) to analyse the A-->G base substitution at nucleotide 3243, 50% of the samples showed detectable levels of that particular mutation, with abundances ranging from 0.01% to 0.12%. In the same set of skin samples, the overall incidence of the 4977 bp "common" deletion was also approximately 50%. Where detected, the abundance of this deletion ranged from 0.0002% to 0.1%. Comparative analyses of the incidence and abundance of these two mutations, collectively and in individual skin samples, led to these two conclusions: (1) there is independent occurrence of these two mtDNA mutations in human skin, and (2) whereas the 4977 bp deletion shows an age-associated accumulation in human skin, no age association is apparent for the 3243 A-->G base substitution. Furthermore, in general, there is a much lower incidence of somatic mutations in mtDNA of human skin as compared to that in postmitotic tissues such as skeletal muscle.