Mitochondrial respiration of adipocytes differentiating from human mesenchymal stem cells derived from adipose tissue.

Burden of obesity is increasing in the contemporary world. Although multifactorial in origin, appropriate mitochondrial function of adipocytes emerges as a factor essential for healthy adipocyte differentiation and adipose tissue function. Our study aimed to evaluate mitochondrial functions of human adipose-derived mesenchymal stem cells committed to adipogenesis. On days 0, 4, 10, and 21 of adipogenesis, we have characterized adipocyte proliferation and viability, quantified lipid accumulation in maturing cells, performed qualitative and quantitative analysis of mitochondria, determined mitochondrial respiration of cells using high-resolution respirometry, and evaluated mitochondrial membrane potential. In the course of adipogenesis, mitochondrial oxygen consumption progressively increased in states ROUTINE and E (capacity of the electron transfer system). State LEAK remained constant during first days of adipogenesis and then increased probably reflecting uncoupling ability of maturing adipocytes. Citrate synthase activity and volume of mitochondrial networks increased during differentiation, particularly between days 10 and 21. In addition, lipid accumulation remained low until day 10 and then significantly increased. In conclusion, during first days of adipogenesis, increased mitochondrial respiration is needed for transition of differentiating cells from glycolytic to oxidative metabolism and clonal expansion of preadipocytes and then more energy is needed to acquire typical metabolic phenotype of mature adipocyte.

Absence of BRAF mutation in pheochromocytoma and paraganglioma.

Pheochromocytomas and Paragangliomas (PHEO/PARA) are rare endocrine tumors originating from the adrenal medulla. More than 20 genes are involved in the tumorigenesis of these tumors, but a substantial part of the causative genetic events remains unexplained. A recent study has reported the presence of the activating BRAF V600E mutation in PCC, suggesting a role for BRAF activation in tumor development. Other studies have not find this mutation. This study investigates the occurrence of the BRAF V600E mutation in these tumors.A cohort of 64 PHEO/PARA were screened for the BRAF V600E mutation using direct Sanger sequencing and QRT-PCR.All cases investigated displayed wild-type without V600E BRAF mutationTaken together with all previously screened tumors up to date, only 1 V600E BRAF mutation has been found among 427 PCCs. These findings imply that the V600E BRAF mutation is a rare event in PHEO/PARA.

New perspectives in diagnosis of gynaecological cancers: Emerging role of circulating microRNAs as novel biomarkers.

Early diagnosis is a prerequisite of the more successful treatment of cancer. In gynaecological cancers, such as ovarian, endometrial and cervical cancers, the recent efforts are aimed at finding novel diagnostic biomarkers to help reduce the worldwide health burden associated with these cancers. In this review, we focus on the recent research progress in circulating, particularly cell-free microRNAs expression achieved in ovarian, endometrial and cervical cancers showing an opportunity to find novel diagnostic biomarkers for these malignant diseases. With the onset of microRNAs investigations showing their diagnostic potential in many diseases, their role in gynaecological cancers has been examined as well. However, similarly as in many other diseases, the vast majority of research on microRNAs expression has been dealing with tissue samples and cell lines. Recently, as the novel approaches focused on cell-free microRNAs expression have emerged, several studies identified their potential diagnostic and prognostic value in gynaecological cancers using blood, serum/plasma or urine samples. More research will be needed to establish circulating and extracellular microRNAs as the novel diagnostic markers for gynaecological malignancies. Inconsistency of results across the studies due to technical and biological variation, and a low number of this kind of investigations are the main potential pitfalls remaining to be resolved.

Clinical relevance of CD157 for rapid and cost-effective simultaneous evaluation of PNH granulocytes and monocytes by flow cytometry.

INTRODUCTION: CD157 has been reported as a potentially useful marker for paroxysmal nocturnal hemoglobinuria (PNH) testing by flow cytometry (FCM). METHODS: We determined the performance characteristics of a CD157-based five-color assay and compared results from patient analysis with results obtained with a previously validated CD14/CD24-based six-color protocol. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analysis of granulocytes ranged from 0.88/0.09% to 3.02/3.71% and from 0.20/0.08% to 8.83/4.04% for the five- and six-color protocol, respectively. For monocyte, CV ranged from 0.42/0.49% to 8.13/4.80% for the five-color protocol and from 0.28/0.70% to 5.41/3.19% for the six-color protocol within various PNH clones. Coefficient of determination (r(2) ) for linear regression analysis of PNH clones ranging from 0.3 to 99.8% was >0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), and Bland-Altman analysis demonstrated agreement with mean bias ranging from -0.02 to 0.38. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, excellent correlation, and agreement between approaches. In agreement with recently published data, our experience supports the clinical relevance of CD157 for a rapid and cost-effective simultaneous evaluation of PNH leukocytes by flow cytometry.

[Differential expression of microRNAs as the novel potential diagnostic tool for the endometrial carcinoma]. / Exprese mikroRNA jako nový potenciální diagnostický nástroj pro karcinom endometria.

The most common gynecological malignancy, the endometrial carcinoma, is mostly diagnosed at early stages. However, diagnosis at advanced stages is accompanied by the high mortality rate. It is suggested that this cancer is one of the less studied female cancers. The necessity to establish novel diagnostic markers has led to investigations of small non-coding RNAs, particularly microRNAs, also in endometrial cancer. There have been found many microRNAs potentially associated with carcinogenesis and clinico-pathological data including prognosis for patients. Many microRNAs may also serve as diagnostic markers for non-invasive diagnostics using blood plasma. We reviewed extensively the published research focused on microRNAs that have been found deregulated particularly in tissue samples within the both major types of endometrial cancer (type 1 and type 2). They are presented in the view of their potential targets and mechanisms of action. Some microRNAs have been found deregulated also in blood plasma. There exists a high level of inconsistency across the studies as many microRNAs have been found only within one or a few studies so far. However, there are some microRNAs consistently deregulated as suggested several investigations. There remains the urgent need of more intensive research focused on the microRNAs and their regulatory role in endometrial cancer. Such a research should provide the basis for the introducing novel diagnostic tools into the clinical practice.

[Cancer stem cells and ovarian cancer Characteristics, importance and potential applications in clinical practice]. / Kmenové bunky a karcinom ovaria Charakteristika, význam a potenciální aplikacev klinické praxi.

Ovarian cancer is the most malignant and the second most common gynecological cancer which encompasses a heterogeneous group of tumors with a different etiology. Extra-ovarian tissues may play a role in the development of ovarian cancer, despite this issue is still debated. This disease is associated with a strong chemoresistance and tendency to recurrence, and asymptomatic behaviour at early stages. Effective screening markers have not been established yet. Cancer stem cells have been postulated to play a role within tumor formation and by the establishment of chemotherapy-resistant population of malignant cells after the surgery and application of chemotherapy. These cells are multipotent cells capable of un-limited divisions and have potential to induce tumorigenesis in immune-suppressed mice. Their detailed characterization is still an open issue, however there have been identified several markers associated with ovarian cancer stem cells. The major markers of ovarian cancer stem cells identified so far are CD133, ALDH, CD44, CD117, CD24 and EpCAM. Their occurrence in primary tumors may be associated with patients´ prognosis; however there are insufficient data for definite conclusions. Dynamic processes of carcinogenesis result also in changes of cancer stem cells phenotypes based on the microenvironmental changes within the tumor. The markers may thus be acquired, or lost, as it has been proven experimentally. As regards the possibility to use targeted, specific therapy approaches, there are some promising studies, suggesting this may be a method of potential treatment. Further characterization and functional studies of cancer stem cells will be necessary for the elucidation of carcinogenesis, chemoresistance and metastases formation-associated processes. Such studies will be the basis for establishment of novel diagnostic, prognostic and therapeutic approaches for the ovarian cancer treatment. The most recent results on ovarian cancer stem cells research are presented in this paper.

[Hereditary pheochromocytoma and paraganglioma]. / Hereditární feochromocytom a paragangliom.

Pheochromocytomas and paragangliomas are tumors arising from chromaffin cells. These tumors produce catecholamines and are typically found with symptoms and signs that may include hypertension (persistent or episodic), palpitations, headache and sweating. So far, 10 different genes have been associated with both tumors and other genes are expected to be detected. Pheochromocytoma and paraganglioma can occur as a part of genetic syndromes - familial paragangliomas (SDH genes, SDHAF2 gene), von Hippel-Lindau syndrome (VHL gene), multiple endocrine neoplasia type 2 (RET gene), and neurofibromatosis type 1 (NF1 gene). These tumors may be the first and only manifestation of these genetic syndromes. Patients with SDHB mutations are at high risk to develop malignant disease and unfortunately current therapeutic options for malignant form of disease are poor. Genetic testing plays a key role in the management of these tumors and therefore not only index patients with pheochromocytoma but also relatives should be tested. Management of this disease requires multidisciplinary cooperation and should be performed in the specialized medical centres.

Ovarian cancer: origin and factors involved in carcinogenesis with potential use in diagnosis, treatment and prognosis of the disease.

Ovarian cancer representing the most lethal gynecologic malignancy escapes from the efforts to manage the disease. We reviewed the current state of the research considering three main concepts on origin of ovarian cancer including epithelial-mesenchymal transition, secondary origin from Müllerian system and cancer stem cell hypothesis. Cytogenetic and molecular characteristics of ovarian cancer are focused particularly on microRNA expression studies revealing huge potential in recent years, although other transcriptomic, proteomic, epigenetic, epidemiologic and immunological factors are touched upon, too. Routine and investigated diagnostic and treatment methods are outlined and several factors revealed to be associated with prognosis of the disease. Despite the huge progress on elucidating factors involved in ovarian cancer carcinogenesis, still remains urgent need to improve both the diagnostics as well as the treatment.

Mutation analysis of the MYH gene in unrelated Czech APC mutation-negative polyposis patients.

Some of the APC negative FAP and AFAP cases have recently been found to be attributable to MYH associated polyposis (MAP). MAP is an autosomal recessive syndrome associated with 5-100 colorectal adenomas and caused by mutation in the MYH gene. Here, we screened for germline MYH mutations in 82 APC-mutation-negative probands with classical and attenuated familial adenomatous polyposis using the denaturing high performance liquid chromatography (DHPLC) method in combination with sequencing. Altogether 12 previously reported changes and four novel genetic alterations, mostly in intronic sequences, were identified. The results revealed the presence of biallelic germline MYH mutations in two patients. These patients were compound heterozygotes for two of the most common germline mutations c.494 A>G (p.Y165C); c.1,145 G>A (p.G382D). These variants are established to be associated with adenomatous polyposis and colorectal cancer. No novel pathogenic mutation has been identified in our study.

[Hereditary forms of colorectal adenomatous polyposis]. / Hereditární formy kolorektální adenomatózní polypózy.

BACKGROUND: Hereditary colorectal adenomatous polyposis syndromes are a predisposition to colorectal carcinoma development. The familial adenomatous polyposis is the most common analyzed syndrome that results from germline mutations in the APC gene. In addition to, the autosomal recessive form of polyposis has been recently reported. This disease is caused by germ-line mutations in the base excision repair MYH gene. The goal of this study is the identification of genetic causes of the colorectal polyposis, the determination of the frequency and type of the APC and MYH germ-line mutations in the set of families with colorectal polyposis in Czech population. METHODS AND RESULTS: The set of 103 probands with FAP was screened for germ-line APC mutations using the Protein Truncation Test and Denaturing Gradient Gel Electrophoresis. The MYH mutational screening was performed on 60 unrelated patients without detected APC mutations using the Denaturing High Performance Liquid Chromatography. Automated sequencing was carried out to identify found mutations. Totally, the 51 germ-line APC mutations (69,9%) are reported in the set of 72 probands including 31 novel mutations unique for Czech population. Molecular genetic analysis of the MYH gene revealed 15 DNA variations (25 %) including two patients identified as p.Y 165C/p.G382D compound heterozygotes (3,3%) and 13 polymorphisms or intronic changes (21,7%). The novel variants were detected in the 5 patients. CONCLUSION: Present study reflects the extremely heterogenous spectrum of the APC mutations in Czech population and confirms the previously reported data. However, the changes found in the MYH gene still need more extensive studies. Our results are important for genetic counselling and further clinical management among at-risk family members. It also enables distinction among different types of the colorectal polyposis.

[Mutations in tumor suppressor gene NBS1 in adult patients with malignancies]. / Mutace v tumor supresor genu NBS1 u dospelých pacientu s malignitami.

BACKGROUND: Mutations 657del5 and R215W in exon 6 of tumor suppressor gene NBS I are found in 1% Slavic populations. Increased occurrence of cancer was repeatedly reported in adult relatives of patients with Nijmegen breakage syndrome. Among children with oncological problematic, nonsignificantly increased frequency of NBS1 heterozygotes was found, which seems not to play any important role in cancerogenesis in childhood. However, the proportion of NBS heterozygotes among adult patients with malignancies could be significant and their therapy and follow up should respect their hyperradiosensitivity. METHODS AND RESULTS: Mutations in exon were studied in 706 adult patients with malignancies. We found 5 NBS heterozygotes, which not more than the population prevalence (1:129-165). Increased frequency of NBS heterozygotes was found among patients with colon and rectal cancer (2/101), breast cancer (1/60), skin malignancies (1/98). CONCLUSIONS: Surprisingly only one NBS heterozygote was found among 228 patients with nonHodgkin lymphoma, the malignancy which is a common complication in NBS homozygotes. Other types of malignancies were uncommon and only one R215W heterozygote was found. Comparison frequency of NBS heterozygotes with incidence NBS among person older than 70 years shows significant difference. Prevention of malignancies by avoidance from ionisation could be realized also in relatives of patients after identification of their genotype.

Molecular analysis of the APC and MYH genes in Czech families affected by FAP or multiple adenomas: 13 novel mutations.

Familial adenomatous polyposis (FAP) is an autosomal dominant predisposition to colorectal cancer and is caused by germline mutations in the adenomatous polyposis coli gene. The most prominent clinical manifestation is the presence of hundreds to thousands of colorectal polyps. A milder phenotype is found in patients affected with AFAP/ multiple adenomas. We screened the entire APC coding region using the combination of DGGE, PTT and direct sequencing and identified causative mutations in 52 of 77 patients. Thirteen of the mutations found were novel. In addition, we also tested 21 APC mutation/negative probands for the two most common mutations in the MYH gene. Four patients showed neither dominant transmission of the disease nor evidence of APC mutations. In one of them the most common biallelic germline mutation in the MYH gene was detected. Correlations between the localization of germline mutations and clinical manifestations of the diseases are discussed.

Familial adenomatous polyposis as a precancerosis of colon cancer.

Familial adenomatous polyposis is a genetic disorder caused by mutations of the adenomatous polyposis coli gene. This gene is localized on chromosome 5q21. The incidence of the disease is 1/7000-8000. Surgery has an important role in the management of familial adenomatous polyposis. In the absence of prophylactic colectomy, death from colom cancer will occur in virtually all of familial adenomatous polyposiscases by age 50, with 37% affected by colon cancer by age 37. (Ref. 8).