A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Primary prevention of cervical cancer: prophylactic human papillomavirus vaccines.

Human papillomavirus (HPV) is one of the most common sexually transmitted diseases worldwide. Cervical and anal intraepithelial neoplasia, genital warts, and recurrent respiratory papillomatosis such as cervical and other anogenital cancers, are HPV-associated diseases. Prophylactic HPV vaccines are composed of HPV L1 capsid protein that self-assembles into virus-like particles (VLPs) when expressed in recombinant systems. The two types of prophylactic vaccines are designed a bivalent vaccine to protect against high-risk HPV types 16 and 18 and a quadrivalent vaccine designed to protect against HPV 16 and 18, and low-risk, genital wart-causing HPV 6 and 11. Proof-of-principle trials have suggested that intramuscular injections of VLPs result in strong adaptive immune responses that are capable of neutralizing subsequent natural infections. Recent research on the safety and efficacy of candidate prophylactic vaccines against HPV have shown very promising results with nearly 100% efficacy in preventing the development of persistent infections and cervical precancerous lesions in vaccinated individuals.

Early complications after nipple-sparing mastectomy and immediate breast reconstruction with silicone prosthesis: results of 214 procedures.

BACKGROUND AND AIMS: breast reconstruction with silicone prosthesis following nipple-sparing mastectomy has become widely accepted as a reconstruction option in women requiring mastectomy for cancer. The purpose of this study was to evaluate the incidence and some factors influencing early local complications in patients undergoing NSM with immediate implant reconstruction. MATERIAL AND METHODS: prospective study was performed on a consecutive series of 214 breast reconstructions in 205 patients. All complications during the six weeks after surgery were recorded. 42 prostheses were implanted after neoadjuvant chemotherapy, 27 patients previously had radiotherapy due to breast conserving surgery and in all other cases surgery was the pri-mary treatment for cancer. RESULTS: the overall six-week complication rate was 16% (35) and included: major skin flap necrosis (4%, 9 procedures), minor skin necrosis (3%, 7), major infection (2%, 5), minor infection (3%, 7), prolonged seroma formation (3%, 6), haematoma (1%, 2) and epidermolysis (1%, 2). In 6% (12) reconstruction procedures explantation of prosthesis was done. Neoadjuvant chemo-therapy and radiotherapy were not associated with higher rate of complications. CONCLUSION: nipple-sparing mastectomy with immediate implant reconstruction has acceptable morbidity rate in the hand of experienced oncoplastic surgeon and therefore should be considered as treatment option to women requiring mastectomy.

Surgical approaches towards fertility preservation in young patients with early invasive cervical carcinoma.

Cervical cancer still remains one of the major problems in developing countries. The last decade of 20th century has seen a trend towards more conservative surgical approaches in the treatment of early-stage disease in young patients. The trend of delaying childbearing, nowadays, increases preservation of fertility, and reproductive function is a major concern when counseling these young women with regard to the effects of treatment for cervical cancer. Radical trachelectomy, either with abdominal or vaginal surgical approach, showed promise as treatment option in young patients with early cervical cancer. The basic principle of such a surgical approach is an operation aiming at preserving the uterine body and removing the cervix, parametrium, with bilateral pelvic lymphadenectomy, and creating a utero-vaginal anastomosis, either laparoscopically or by laparotomy. Both surgical approaches are evaluated after a search of the relevant literature in Pub Med or Medline.

Linear trend analysis of patients with cervical cancer treated at the Institute of Oncology Vojvodina in 2001-2007.

PURPOSE: Cervical cancer is the leading cause of death in women with gynecological cancers in Vojvodina. Serbia currently holds the leading place in Europe regarding the incidence of cervical carcinoma and comes second in terms of mortality. METHODS: Data were retrieved from the Register for malignant neoplasms of the Institute of Oncology Vojvodina for the period 2001-2007. The patients were divided in 3 groups according to the stage of disease based on the FIGO classification for cervical cancer. Data were analysed using linear trend and t-test. RESULTS: The linear trend of the number of registered cases in the group of stage I-IIA patients during 7 years showed no significant change in the prevalence of the disease. In the group of women diagnosed with stage IIB no statistical difference regarding either a rising or a decreasing trend was observed. The observed trend in an advanced disease stage (III and IV) showed a slight decrease in the number of patients, but without statistical significance. CONCLUSION: The linear trend of the number of patients with cervical carcinoma during a 7-year period points to the fact that the number of newly detected cases of advanced disease stages did not decrease significantly despite the affordable and simple methods of early detection. This result underlines the importance of implementation of a National screening programme in the general population for early detection of cervical neoplasms. The excellent results of National screening programmes in other European countries lend support to this approach.

Clinical staging and histopathological findings after radical hysterectomy in FIGO stage IIB cervical cancer.

PURPOSE: Advanced cervical cancer still represents a major health care challenge in the developing world. According to standard protocols the treatment of choice for stage IIB cervical cancer is cisplatin-based chemoradiotherapy. However, in some European countries, and especially in Japan, patients with stage IIB cervical cancer are generally treated with radical hysterectomy as initial treatment. The aim of this study was to compare clinical stage with pathological findings, and also to correlate any relationship between parametrial infiltration and nodal status. PATIENTS AND METHODS: From 1997 to 2006, 26 patients with FIGO stage IIB cervical cancer were radically operated (Piver class III operation). Preoperative clinical findings were compared with the pathological findings of the surgical material. The correlation between infiltration of the parametria and lymph node status was also examined. Fisher's exact test was used to examine statistical significance. RESULTS: The patients' median age was 48.3 years (range 36-61). The median number of removed lymph nodes was 16 (range 8-40). The histopathological types of cervical tumors were: squamous cell carcinoma 80%, adenosquamous carcinoma 15% and adenocarcinoma 5%. In 50% of the patients the parametria were infiltrated, suggesting that 50% of the patients were clinically overstaged. Positive lymph nodes were found in 69% of patients with positive parametria and 15% in patients with negative parametria (p <0.05). Patients with positive lymph nodes received adjuvant chemoradiotherapy. CONCLUSION: Adequate preoperative staging such as clinical examination under anesthesia or nuclear magnetic resonance could help to exclude parametrial involvement in equivocal cases. Parametrial invasion presents an important risk factor for lymph nodes metastases.

Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner.

In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway.

c-Jun N-terminal kinase 2 promotes enterocyte survival and goblet cell differentiation in the inflamed intestine.

c-Jun N-terminal kinases (JNKs) contribute to immune signaling but their functional role during intestinal mucosal inflammation has remained ill defined. Using genetic mouse models, we characterized the role of JNK1 and JNK2 during homeostasis and acute colitis. Epithelial apoptosis, regeneration, differentiation, and barrier function were analyzed in intestinal epithelium-specific (ΔIEC) or complete JNK1 and bone marrow chimeric or complete JNK2 deficient mice as well as double-knockout animals (JNK1ΔIECJNK2-/-) during homeostasis and acute dextran sulfate sodium (DSS)-induced colitis. Results were confirmed using human HT-29 cells and wild-type or JNK2-deficient mouse intestinal organoid cultures. We show that nonhematopoietic JNK2 but not JNK1 expression confers protection from DSS-induced intestinal inflammation reducing epithelial barrier dysfunction and enterocyte apoptosis. JNK2 additionally enhanced Atonal homolog 1 expression, goblet cell and enteroendocrine cell differentiation, and mucus production under inflammatory conditions. Our results identify a protective role of epithelial JNK2 signaling to maintain mucosal barrier function, epithelial cell integrity, and mucus layer production in the event of inflammatory tissue damage.

Lung cancer trends in Vojvodina.

PURPOSE: Lung cancer in Vojvodina is the leading form of cancer as well as the leading cause of cancer deaths in men. It ranks 2nd in cancer deaths in women in Vojvodina. The goal of this report was to study lung cancer in Vojvodina in th period 1989-1998 by analysing the crude and age-standardized incidence and mortality rates in the male and female population. MATERIALS AND METHODS: Data used for analyses were provided by the Cancer Registry of Vojvodina from the Institute of Oncology Sremska Kamenica. Data included number of cases and deaths of lung cancer, by age groups in 5 year intervals and by the municipalities for male and female population separately. Descriptive epidemiological method was used. RESULTS: An increasing tendency of linear trend of lung cancer incidence in males based on crude and age-standardized incidence rates was found. The trend of lung cancer incidence in males was highly intense and extremely unfavorable. In females, an increasing tendency of linear trend of lung cancer incidence based on crude and age-standardized incidence rates was also found; this was not as intense but it was unfavorable. An increasing tendency of linear trend of lung cancer mortality based on crude and age-standardized mortality rates in both sexes was registered, which was not intense, but it was unfavorable. CONCLUSION: Vojvodina is a region with high incidence and mortality rates in comparison to neighboring and European Union (EU) countries both in male and female population.

Clinical-sonographic scoring system in noninvasive diagnosis of endometrial cancer.

PURPOSE: In 90% of all endometrial cancers vaginal bleeding is the leading clinical symptom. Nowadays, scoring systems have become acceptable in medicine as less invasive, adequate, diagnostic methods. The main goal of this study was to examine the clinical-sonographic scoring system as a noninvasive diagnostic method for endometrial cancer. PATIENTS AND METHODS: The study included 122 patients with postmenopausal bleeding (PMB). Transvaginal sonography was performed before curettage. Patients were divided in two groups (A and B). In group A included were patients without endometrial malignancy and in group B were patients with endometrial cancer. A clinical-sonographic scoring system named ONCO 1 was created. Each patient got her own score based on anamnesis, clinical examination, and transvaginal ultrasonography. Evaluations of the clinical-sonographic scoring system were performed by using the test for diagnostic accuracy and receiver operating characteristic (ROC) curve. RESULTS: Patients with endometrial cancer were older (median age in group B 64.49 years vs. 58.81 in group A), the length of corpus uteri was longer (6.41 cm in group B vs. 5.25 cm in group A), and the postmenopausal period was longer (13.67 years median in group B vs. 9.11 in group A). All parameters were statistically significant. The average value of clinical-sonographic scoring system ONCO 1 in group A was 7.13, +/-3.07 SD and in group B it was 9.14, +/-2.32 SD. The difference was statistically significant. CONCLUSION: Postmenopausal bleeding caused by endometrial cancer is usually diagnosed in older patients. It was possible to distinguish high-risk patients with neoplasia from those with benign changes of the endometrium using the clinical-sonographic systems ONCO 1. Nevertheless, histopathological examination is still unavoidable for the final diagnosis of endometrial cancer.

Regulation of lipoprotein lipase by glucose in primary cultures of isolated human adipocytes. Relevance to hypertriglyceridemia of diabetes.

Human adipose tissue lipoprotein lipase (LPL) is stimulated in vivo by an insulin-glucose infusion. However, previous work by us showed no effect of physiologic insulin concentrations on LPL in isolated human adipocytes. To pursue further the regulation of LPL in vitro, primary cultures of isolated human adipocytes were prepared and exposed to glucose concentrations of 0-4.5 mg/ml. LPL activity was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests (EXT). After 5 h in culture, a stimulatory effect of glucose on HR was observed. After 24 h there was a gradual increase in CM, HR, and EXT in parallel with increasing glucose concentrations of 0-1.0 mg/ml. At glucose concentrations greater than 1.0 mg/ml, however, there was a decrease in CM. At a glucose concentration of 4.5 mg/ml, CM was only 51 +/- 14% (P less than .02) of its value at glucose concentrations of 1.0 mg/ml. Cellular LPL (HR and EXT) was not affected by high glucose concentrations. Response of cellular LPL to the hormonal regulator insulin-like growth factor I (IGF-I) was modulated by medium glucose. HR in cultures treated with 50 ng/ml IGF-I was 166 +/- 40 and 147 +/- 23% of HR in control cultures at glucose concentrations of 1.0 and 2.5 mg/ml, respectively (P less than or equal to .05). However, IGF-I failed to stimulate HR at glucose concentrations greater than 2.5 mg/ml or less than 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage IB2 cervical cancer: brachytherapy followed by radical hysterectomy.

PURPOSE: Stage IB2 squamous cell cervical cancer can be treated by radiation therapy alone or by radical hysterectomy and lymphadenectomy (pelvic-/+para-aortic). Preoperative radiation therapy followed by extrafascial hysterectomy has been recommended as an effective combined treatment method. PATIENTS AND METHODS: During the period January 1994-January 2004, 114 patients with stage IB2 cervical cancer were treated with preoperative brachytherapy followed by radical hysterectomy (Piver class III) with pelvic lymphadenectomy. RESULTS: Histology showed that 56 (49%) patients were without cervical malignant disease. Positive lymph nodes were found in 5 (9%) of them and negative in 51 (91%). In 58 (51%) patients cervical cancer still existed after brachytherapy and among them 26 (45%) were with lymph node metastasis. Patients with residual cervical carcinoma and positive lymph nodes after brachytherapy were older than those with no residual carcinoma and negative lymph nodes. CONCLUSION: Women with stage IB2 squamous cell cervical cancer primarily treated with brachytherapy must be assessed by appropriate diagnostic procedures to evaluate local effects of brachytherapy and the status outside the pelvis. Negative local findings with positive lymph nodes point to further treatment of patients, while positive local findings point to radical surgery which may increase recurrence-free interval.

The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin-induced apoptosis.

The regulation of apoptosis is believed to be dependent on the balance of the activities of different intracellular signalling systems. Activation of the SAPK/JNK pathway is implied in pro-apoptotic signalling, while activation of the MEK1/ERK pathway may have a viability-promoting effect. We show here that treatment with the MEK1 inhibitor PD98059 sensitizes the human melanoma cell line C8161 to cisplatin-induced apoptosis. In these cells, cisplatin at 40 microM did not elicit significant cell death, whereas massive cell death was seen when cells were pretreated for 20 h with 40 microM PD98059 before the addition of cisplatin. Concomitant addition of PD98059 and cisplatin did not have any sensitizing effect, and PD98059 on its own did not induce apoptosis. However, in three other human melanoma cell lines PD98059 did not potentiate cisplatin-induced apoptosis. Instead, in one of these cell lines (AA), PD98059 protected against cisplatin-induced cytotoxicity. We conclude that blocking of the MEK1/ERK pathway may, in some instances, potentiate the cytotoxic effect of cisplatin on human melanoma cell lines, whereas in other instances it may have a protective effect. Thus it cannot be regarded as a general approach to sensitizing melanoma cells to drug-induced apoptosis.

Paclitaxel or docetaxel combined with platinum in advanced ovarian cancer?

Epithelial carcinoma of the ovary is one of the most common gynecologic malignancies and ranks 5th as a cause of cancer death in women, with half of all cases occurring in women aged over 65. Because ovarian cancer is often asymptomatic in its early stages, most patients have widespread disease at the time of diagnosis. Surgery is required for correct diagnosis and accurate staging, representing also the main form of treatment for early-stage disease which is confined to the ovaries. In advanced stages (III and IV) where the tumor has spread beyond the pelvis, the initial surgical treatment is followed by chemotherapy. Ovarian tumors are sensitive to chemotherapy, and most stage III and IV patients receive chemotherapy to increase survival, disease-free interval and improve quality of life (QoL). Until the mid 1990s standard chemotherapy of advanced ovarian cancer involved a platinum compound, either cisplatin or carboplatin, administered either alone or in combination with cyclophosphamide. New therapy standards emerged after the publication of a trial by the Gynecologic Oncology Group ( GOG) in 1996 and the first reports of an intergroup trial in 1998 that confirmed the GOG results. These studies demonstrated that patients treated with cisplatin/paclitaxel had significantly higher response rates, progression-free survival and overall survival compared with the previous standard treatment of cisplatin plus cyclophosphamide.

Tumor angiogenesis in gynecological oncology.

Angiogenesis, the formation of new blood vessel networks to permit sustained tumor growth, is one of the most rapidly growing fields in basic and applied cancer research. Angiogenesis is a prerequisite for tumor growth and metastasis. Vascular endothelial cell proliferation, migration and capillary formation are stimulated by angiogenic growth factors. It is now accepted, mainly on the basis of knock-out experiments, that the most potent pro-angiogenic factor is the vascular endothelial growth factor (VEGF). VEGF is the prototype of a family of pro-angiogenic peptides, which now comprises several related members. Recent studies in gynecological oncology have shown considerable importance of tumor angiogenesis as independent prognostic factor, or the use of angiogenesis-related factors as possible tumor markers. Immunohistochemical studies have revealed that high microvessel density (MVD) was associated with poor prognosis in carcinomas of the uterine cervix, endometrium, vulva and ovary. Tumor angiogenesis is not a specific process for one type of tumor, and angiogenic growth factors cannot be specific for one type of malignant disease. Immunohistochemical studies also showed importance of angiogenic factors and MVD in other kinds of cancers such as breast cancer, gastrointestinal cancers, soft-tissue sarcomas etc. The importance of tumor angiogenesis in the natural history of cancer, the possible applications of angiogenesis markers as prognostic factors and the emergence of innovative antitumor treatments based on anti-angiogenic strategies may lead to new goals in oncology.

Retroperitoneal malignant schwannoma and peritoneal malignant mesothelioma: a case report.

Malignant schwannoma and peritoneal malignant mesothelioma (MM) are very rare tumors. Schwannoma or neurilemmoma-benign or malignant-do not arise from the nerves, but from the supporting Schwann cells. Malignant peripheral nerve sheath tumors (MPNSTs) commonly are large in size. They are painful and may cause many different symptoms depending on their location and size. MM arises primarily from the surface serosal cells of the pleural, peritoneal, and pericardial cavities. A malignant schwannoma was diagnosed in a 52-yearold woman, which was surgically treated. After a 17-month disease-free interval, local recurrence was diagnosed. The patient was operated on for second time and MM was diagnosed as second primary tumor, along with recurrence of the malignant schwannoma. The patient received postoperative adjuvant external beam radiotherapy and chemotherapy. Despite combined-modality treatment the disease progressed and the patient was operated on for third time 2.5 years after the first operation with partial tumor resection. She died 3 years after the first diagnosis. Early diagnosis of these two types of tumors is very difficult because of unspecific clinical symptoms. Singlemodality therapy of these tumors has shown poor results. Combined-modality approaches have shown some benefits, but further studies are required.

Benign and malignant struma ovarii: report of three cases and review of the literature.

Struma ovarii belongs to the group of monodermal and highly specialized teratomas and comprises less than 5% of mature teratomas. Thyroid tissue is present exclusively or predominantly. Malignant transformation in struma ovarii is uncommon, and when present, it exhibits a follicular pattern most of the times. Three patients with the diagnosis of struma ovarii are presented herein. Two of them had a unilateral adnexal mass with ascites in one case. Total abdominal hysterectomy with bilateral salpingo-oophorectomy and omentectomy were carried out in both patients. In both patients thyroid hormonal status was normal pre and postoperatively. The third patient had a histology compatible with follicular carcinoma of the thyroid tissue of struma ovarii; strumal carcinoid was also present. Postoperatively monochemotherapy with etoposide was administered for 6 cycles every 3 weeks. Thyroid hormones and thyroid ultrasonography (US) were also normal in this patient. Surgical management is the treatment of choice for struma ovarii which also represents a preventive measure for possible future malignant transformation.

Radical hysterectomy for stage IB1 cervical carcinoma: lymph node metastasis as a prognostic factor.

PURPOSE: Lymph node metastasis is one of the most important factors influencing prognosis and further therapy in patients with cervical carcinoma. The aim of this study was to confirm the impact of nodal metastasis od disease-free interval in women with stage IB1 cancer of the uterine cervix. PATIENTS AND METHODS: From June 1986 to December 1999 269 patients with stage IB1 cervical carcinoma were operated on. Two hundred thirty-six (87.84%) patients had class III and 33 (12.16%) class II radical hysterectomy, according to Piver's classification. RESULTS: The median number of the removed lymph nodes was 21. Positive lymph nodes were found in 71 (25.28%) patients. All patients with positive lymph nodes received postoperative adjuvant external beam radiotherapy. Patients with bulky nodal disease received also chemotherapy.Overall 5-year disease-free interval in 212 patients was 80%. Five-year disease-free interval for patients without lymph node metastasis was 91%, while it was 40% in those with lymph node metastasis (p < 0.0001). CONCLUSION: Surgical staging of cervical cancer, which includes pelvic and para-aortic lymphadenectomy,together with pathological data, can provide potentially useful information for the radiation oncologist and precise analysis of survival and prognostic risk factors.

Effects of cadA gene on cell division phenotype.

Studying cell division regulatory mechanisms in Escherichia coli an important role of cad operon was discovered. The cadA gene is part of the cad operon. The promotor of cad operon (pBA) regulates two genes, cadB (the first gene in the cad operon) and cadA (the second one). The cadB encodes the lysine-cadaverine antiporter. The cadA gene encodes enzyme lysine decarboxylase that turns lysine into cadaverine and carbon dioxide. The expression of cadA is activated by several different environmental parameters, such as low pH, low oxygen, and excess lysine. Regulation of any of these parameters depends on the presence of cadC gene, encoding the regulator of the operon. It is located upstream of cad operon. The aim of this study is to investigate if the effect upon the cell division rate was caused by the induction of cadA. Specific aims were to obtain structural gene of cadA and construct a plasmid (pAM1) that contains cadA under the control of the arabinose inducibile promoter pARA, and complement the cell division phenotype of a cadA mutant by providing cadA on the inducibile plasmid. Activation of the pAM1 with 0.05% arabinose reduced the cell division rate that confirms the inhibitory effect of the pAM1.