Evidence that Proteasome-Dependent Degradation of the Retinoblastoma Protein in Cells Lacking A-Type Lamins Occurs Independently of Gankyrin and MDM2

A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a)-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf). p14(arf) expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells.Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to p14(arf)-mediated cell cycle arrest, as was previously shown with p16(ink4a). Potential roles of lamin A/C in the suppression of tumorigenesis are discussed

Potent Anti-Tumor Effect Generated by a Novel Human Papillomavirus (HPV) Antagonist Peptide Reactivating the pRb/E2F Pathway

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein believed to play a major role in cervical cancer. In this study, an antagonist peptide against HPV16E7 protein was first identified from screening the c7c phage display peptide library. The binding specificity and affinity of the selected peptide to HPV16E7 were tested by competitive enzyme-linked immunosorbent assay (ELISA). The antagonist peptide showed obvious anti-tumor efficacy both in cell lines and animal tumor models. Significant cell proliferation inhibition with high specificity was noted when HPV16-positive cells were treated with the peptide. This anti-tumor efficacy was resulted from overriding the activities of HPV16E7 and reactivating the pRb/E2F pathway, as shown by a series of experiments. Flow cytometry analysis revealed that the selected peptide induced G1 arrest in a dose-dependent manner. Competitive ELISA, pull down, and Co-IP experiments indicated that the selected peptide disrupted the interaction between HPV16E7 and pRb proteins both in vitro and in vivo. Luciferase reporter assay verified that transcription activities of E2F were suppressed by the peptide through restoration of pRb. RT-PCR and Western blot revealed that it reduced cyclins A, D1, and E1 expression, and led to HPV16E7 protein degradation, but pRb protein stabilization. The current study suggests that this specific peptide may serve as a potential therapeutic agent for HPV16-positive cervical cancer

PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein

The retinoblastoma protein (RB)–E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G1/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB–E2F1 pathway

Efficacy of <i>Helicobacter pylori</i> eradication therapy of infection with omeprazole and rabeprazole in overweight and obese patients

Введение. В ряде исследований была выявлена достоверная ассоциация между низкой эффективностью эрадикационной терапии инфекции Helicobacter pylori (H. pylori) и избыточной массой тела или ожирением у пациентов без сахарного диабета. Данный тренд может объясняться различиями в профиле фармакокинетики ингибиторов протонной помпы, входящих в состав схем эрадикации.Цель исследования: оценить эффективность эрадикационной терапии инфекции (H. pylori) при использовании препаратов омепразола и рабепразола у пациентов с избыточной массой тела и ожирением.Материалы и методы: в проспективное рандомизированное сравнительное исследование было включено 34 пациента (21 мужчина и 13 женщин) с H. pylori -ассоциированной патологией и избыточной массой тела или ожирением (согласно классификации ВОЗ). В процессе рандомизации пациентов было сформировано 2 равные группы в зависимости от назначаемой схемы ЭТ. Первая группа (n = 17) получала классическую тройную схему ЭТ с омепразолом (омепразол 20 мг х 2 раза в стуки, амоксициллин 1000 мг х 2 раза в стуки, кларитромицин 500 мг х 2 раза в сутки) в течение 10 дней. Второй группе (n = 17) назначалась классическая тройная схема ЭТ с рабепразолом (рабепразол 20 мг х 2 раза в стуки, амоксициллин 1000 мг х 2 раза в сутки, кларитромицин 500 мг х 2 раза в сутки) в течение 10 дней. Скрининг и контроль эрадикации инфекции H. pylori производились при помощи быстрого уреазного теста биоптата или С13-уреазного дыхательного теста. Оценка эффективности ЭТ осуществлялась не ранее чем через 6 недель после окончания курса лечения.Результаты: Средний возраст пациентов составил 41,9 лет (95% ДИ: 36,5-47,2), а медиана ИМТ - 28,1 кг/м2 (95% ДИ: 26,9-32,0). Эффективность ЭТ в первой группе составила 64,7% (ITT), 73,33% (PP), а во второй группе - 82,35% (ITT), 87,5% (PP). Использование рабепразола в схеме классической тройной ЭТ потенцирует эффективность лечения у пациентов с избыточной массой тела и ожирением в выборке ITT (ОШ 2,54; 95% ДИ: 0,51-12,54) и PP (ОШ 2,54; 95% ДИ: 0,39-16,55) в сравнении с применением омепразола. При отборе для анализа исключительно пациентов с ожирением (ИМТ ≥ 30 кг/м2) в выборке PP эффективность ЭТ в первой группе составила 50,0%, а во второй - 83,33%.Заключение: проведенное исследование и данные литературного анализа позволяют сделать вывод, что тактика оптимизации ЭТ инфекции H. pylori с использованием рабепразола в качестве ИПП является наиболее многообещающей стратегией повышения эффективности лечения, особенно в группах риска неэффективного лечения, к которым относятся пациенты, страдающие избыточной массой тела и ожирением

A case of a 13-year-old patient with whipple disease

In the present article, the authors provide detailed material on the 13-year observation of a patient with such a rare disease as Whipple’s disease. Pathophysiological aspects, in particular changes in immunity within the framework of this nosology, clinical picture are also described in detail, and the emphasis is placed on the interrelation of certain symptoms, their intensification and regression. Taking into account the absence of clear recommendations for the management of such patients, the article considers various options of therapeutic schemes, and also reflects the own results of treatment

Primary biliary cholangitis – a new nosological unit in the classification of liver diseases (literature review and own clinical observation)

Identification of changes in biochemical parameters of liver functional activity during screening studies requires additional examination of the patient in order to determine the genesis of the disease. In recent years, in routine practice, the most frequently used is an isolated definition of the level of transaminases (ALT, AST), which does not allow timely detection of latent cholestasis syndrome. Primary biliary cholangitis (PBC), previously referred to as primary biliary cirrhosis, is a relatively rare chronic autoimmune cholesthetic liver disease, predominantly affecting middle-aged women and prone to progressing liver cirrhosis. The recommendations of AASLD and EASL note the need for long-term monitoring of patients with ongoing UDCA therapy and regular diagnostic studies to identify signs of disease progression. A clinical example of successful treatment of a patient with PBC with the Russian drug Exhol® is described

Difficulties of differential diagnosis of portal hypertension: case report

In the article we analysed the difficulties of differential diagnosis of portal hypertension, considers a clinical case that illustrates the presented theoretical material. In the presented clinical observation, the patient’s disease was manifested by bleeding from the varicose veins of the esophagus. In most cases, portal hypertension syndrome in practicing clinicians is associated with liver cirrhosis, however, it is necessary to remember about the possibility of developing subhepatic portal hypertension, in particular as a result of the formation of portal vein thrombosis. If there are signs of portal hypertension, it is necessary to specify the level of obstruction to blood flow, that is, the form of portal hypertension (subhepatic, hepatic, suprahepatic). Often, portal vein thrombosis can be formed due to undiagnosed blood diseases that occur without any clinical symptoms. The provided clinical example demonstrates a case of portal hypertension in the outcome of a chronic form of myeloproliferative syndrome. Portal cavernoma is quite rare and it is formed due to multiple small-diameter venous structures that gradually replace the occluded vessel with a system of collaterals proximal and distal to the portal vein thrombosis site. In the formation of the diagnosis the main are radiation research methods, but the conclusions should be considered only in conjunction with the clinical evidence. The clinical case is interesting because a large cavernoma of the portal vein in a patient with subhepatic portal hypertension was regarded as a «solid formation» according to magnetic resonance tomography. According to the literature data, cavernous transformation has an external similarity to the tumor process, which expands the range of differential diagnosis and requires the exclusion of oncological formations

Analysis of the incidence of malignant lymphoma in Kursk areas from 2007 to 2014

Based on incidence data malignant lymphomas in Russia, Central federal district and in the Kursk region from 2007 to 2014. Analyzed the incidence of malignant lymphomas in Kursk region in the overall structure of morbidity malignant neoplasms in Russia and Central federal district in that period, and made disease forecast malignant lymphoma 6 years (up to 2020) in Kursk region

Epstein–Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase

Epstein–Barr virus (EBV) stimulates the proliferation of latently infected B cells and promotes lymphoid malignancies in humans. To address the role of EBV latency protein Epstein–Barr nuclear antigen 3C (EBNA3C) in regulation of the retinoblastoma protein (Rb), we transfected EBNA3C into 293, BJAB, and SAOS-2 cells. In this context, a dominant effect of EBNA3C is to decrease Rb protein levels. EBNA3C also rescues an Rb-induced flat cell phenotype and targets Rb for proteasome- and ubiquitin-dependent degradation. Further, EBNA3C forms a stable complex with Rb in cells when the proteasome machinery is inhibited and interacts with Rb in vitro, mapping to a conserved domain at the terminus of EBNA3C. Deletion analysis of EBNA3C identified a motif within amino acids 140–149 important for both the binding and regulation of Rb. This motif is of particular interest, because it has also been linked to regulation of the Skp1/Cul1/F-box complex, SCF(Skp2). Indeed, inhibition of Skp2 function with a dominant-negative molecule reduces the ability of EBNA3C to degrade Rb. Skp2 has no detectable effect on Rb levels in the absence of EBNA3C, suggesting that SCF(Skp2) is specifically usurped by EBNA3C for the enhancement of Rb degradation. That EBNA3C has exploited this association suggests that other human malignancies might use a similar strategy to regulate the Rb protein