Oropharyngeal cancer related to Human Papilloma Virus: incidence and prognosis in Madrid, Spain

PURPOSE: To report the incidence of HPV-related oropharyngeal cancer (OC) in our region, and determine the influence of HPV status on survival among patients treated with chemoradiation (CRT). METHODS: A total of 102 patients with stage II-IV OC treated by CRT at four hospitals in Madrid, Spain were retrospectively reviewed. Immunohistochemistry analysis was performed to evaluate p16 expression in pretreatment tumor block samples obtained from these patients. HPV-positive and HPV-negative patients were compared to assess differences in overall survival (OS), loco-regional control and disease-free survival. RESULTS: Of the tumor samples evaluated, 26.7 % were p16 positive. HPV-positive patients were younger (median age, 56 vs 59 years; p = 0.052). No significant differences were observed in terms of tumor stage, gender, or smoking habit between HPV+ and HPV- patients. HPV+ patients showed a trend towards better OS (67.4, vs 49.7 %; hazard ratio, 0.55; p = 0.095). CONCLUSIONS: Incidence of HPV-related OC in our region is similar to that reported in other regions in Europe, yet lower than in North America. We observed a trend for improved OS in patients with HPV+ oropharyngeal cancer (AU)

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Oropharyngeal cancer related to Human Papilloma Virus: incidence and prognosis in Madrid, Spain.

PURPOSE: To report the incidence of HPV-related oropharyngeal cancer (OC) in our region, and determine the influence of HPV status on survival among patients treated with chemoradiation (CRT). METHODS: A total of 102 patients with stage II-IV OC treated by CRT at four hospitals in Madrid, Spain were retrospectively reviewed. Immunohistochemistry analysis was performed to evaluate p16 expression in pretreatment tumor block samples obtained from these patients. HPV-positive and HPV-negative patients were compared to assess differences in overall survival (OS), loco-regional control and disease-free survival. RESULTS: Of the tumor samples evaluated, 26.7 % were p16 positive. HPV-positive patients were younger (median age, 56 vs 59 years; p = 0.052). No significant differences were observed in terms of tumor stage, gender, or smoking habit between HPV+ and HPV- patients. HPV+ patients showed a trend towards better OS (67.4, vs 49.7 %; hazard ratio, 0.55; p = 0.095). CONCLUSIONS: Incidence of HPV-related OC in our region is similar to that reported in other regions in Europe, yet lower than in North America. We observed a trend for improved OS in patients with HPV+ oropharyngeal cancer.

Situação epidemiológica da brucelose bovina no Estado de Rondônia / Epidemiological situation of bovine brucellosis in the State of Rondônia, Brazil

Realizou-se um estudo para caracterizar a situação epidemiológica da doença no Estado de Rondônia. O Estado foi estratificado em três circuitos produtores. Em cada circuito produtor foram amostradas aleatoriamente cerca de 300 propriedades e, dentro dessas, foi escolhido, de forma aleatória, um número pré-estabelecido de animais, dos quais foi obtida uma amostra de sangue. No total, foram amostrados 9.717 animais, provenientes de 927 propriedades. Em cada propriedade amostrada foi aplicado um questionário epidemiológico para verificar o tipo de exploração e as práticas zootécnicas e sanitárias que poderiam estar associadas ao risco de infecção pela doença. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e o reteste dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo, se pelo menos um animal foi reagente às duas provas sorológicas. As prevalências de focos e de animais infectados do Estado foram de 35,2 por cento [32,1-38,4 por cento] e 6,2 por cento [4,9-7,6 por cento], respectivamente. Os resultados para os circuitos pecuários foram: circuito 1, 41,9 por cento [36,3-47,6 por cento] e 8,3 por cento [5,9-10,8 por cento]; circuito 2, 31,7 por cento [26,5-37,2 por cento] e 5,9 por cento [4,3-7,6 por cento]; circuito 3, 31,9 por cento [26,7-37,4 por cento] e 4,6 por cento [2,5-6,6 por cento]. Os fatores de risco (odds ratio, OR) associados à condição de foco foram: histórico de aborto (OR= 1,42 [1,04-1,95]) e exploração de corte (OR= 1,75 [1,30-2,38]).

A study to characterize the brucellosis epidemiological situation in the State of Rondônia was carried out. The State was divided into three regions. Three hundred herds were randomly sampled in each region. A pre-established number of animals were sampled in each of these herds. From 927 herds and 9,717 serum samples were collected. In each herd, it was applied an epidemiological questionnaire regarding herd features and also husbandry and sanitary practices that could be associated with risk of infection. The serum samples were screened for antibodies to Brucella spp. by the Rose-Bengal Test (RBT), and all RBT-positive sera were re-tested by the 2-mercaptoethanol test (2-ME). The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalences of infected herds and animals in Rondônia State were 35.2 percent [32.1-38.4 percent] and 6.2 percent [4.9-7.6 percent], respectively. In the regions, the prevalences of infected herds and animals were, respectively: region 1, 41.9 percent [36.3-47.6 percent] and 8.3 percent [5.9-10.8 percent]; region 2, 31.7 percent [26.5-37.2 percent] and 5.9 percent [4.3-7.6 percent]; and region 3, 31.9 percent [26.7-37.4 percent] and 4.6 percent [2.5-6.6 percent]. The risk factors (odds ratio, OR) associated with the presence of the infection were: recent history of abortion (OR= 1.42 [1.04-1.95]) and beef herd (OR=1.75 [1.30-2.38]).

A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors.

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.

Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents.

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.

Amino acid substitutions that convert the protein substrate specificity of farnesyltransferase to that of geranylgeranyltransferase type I.

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Advances in the development of farnesyltransferase inhibitors: substrate recognition by protein farnesyltransferase.

A variety of compounds that show promise in cancer chemotherapy and chemoprevention have been identified as farnesyltransferase inhibitors. These can be classified into mainly two different types of inhibitors, farnesyl diphosphate competitors and CAAX peptidomimetics. The former type acts by competitively inhibiting farnesyltransferase with respect to one of the substrates, farnesyl diphosphate, whereas the latter type acts by mimicking the other substrate, the C-terminal CAAX motif of Ras protein. One example of a farnesyl diphosphate competitor is manumycin, an antibiotic detected in the culture media of a Streptomyces strain. The CAAX peptidomimetics were developed based on the unique property of farnesyltransferase to recognize the CAAX motif at the C-terminus of the protein substrate. Our recent studies have focused on understanding the structural basis of this CAAX recognition. By using in vitro mutagenesis, residues of yeast farnesyltransferase important for the recognition of the CAAX motif have been identified. Two of these residues are closely located at the C-terminal region of the beta-subunit of farnesyltransferase. These and other results on the structural basis of the CAAX recognition may provide information valuable for structure-based design of farnesyltransferase inhibitors.

Kir, a novel Ras-family G-protein, induces invasive pseudohyphal growth in Saccharomyces cerevisiae.

Kir belongs to a novel class of Ras-family G-proteins which includes Gem and Rad. These proteins are unique among Ras super-family G-proteins since their expression is under transcriptional regulation in mammalian cells. To gain insight into the function of Kir, we took advantage of the well-defined signal transduction pathways of yeast. When kir is expressed in Saccharomyces cerevisiae, the transformants form pseudohyphae and exhibit invasive properties characteristics of yeast cells undergoing a developmental transition induced by nitrogen starvation. Analysis of pseudohyphal signaling pathway mutants suggests that the Kir-induced pseudohyphae formation requires a MAP kinase cascade involving ste20, ste11, ste7 but not ste5 gene products. Furthermore, our results are consistent with the idea that Kir functions upstream of the STE20 kinase which plays a critical role in two distinct MAP kinase cascades.