Caracterização imunocitoquímica da expressão da proteína RAP1 em blocos de células escamosas provenientes de citologia cervical em meio líquido

O diagnóstico precoce acurado do câncer cervical, relevante problema de saúde pública no mundo e no Brasil, pela citologia oncótica (Teste de Papanicolaou), é muito prejudicado pela subjetividade e variabilidade dos resultados falsos negativos e falsos positivos do método, particularmente diante de células escamosas atípicas(ASC). Recentes inovações técnicas, como citologia em meio líquido e imunocitoquímica com biomarcadores de proliferação celular, aumentaram a expectativa de melhorias no rastreamento do câncer cervical. No entanto, a aplicabilidade destas inovações nos estágios mais iniciais de atipia e displasias epiteliais permanecem incertas. Assim, considerando resultado prévio do nosso grupo de pesquisa, que identificou a proteína RAP1 como biomarcador diagnóstico da displasia cervical, este trabalho tem como objetivo caracterizar a expressão da proteína RAP1, comparativamente à expressão dos biomarcadores p16 e Ki-67, por imunocitoquímica, em blocos de células escamosas cervicais para possível aplicabilidade na triagem do câncer do colo do útero. Para tal, 34 amostras, 27 pacientes com diagnóstico de alterações celulares benignas (ACB) e 7 pacientes com diagnóstico de ASC foram coletadas na unidade Jenny Faria do Hospital das Clínicas da UFMG. Os resultados indicaram que 85% das amostras de blocos celulares foram satisfatórias para análise morfológica e a técnica reproduziu os principais parâmetros citopatológicos da citologia convencional.

Em relação a sua utilização para diagnóstico, o bloco de células apresentou sensibilidade de 38,46%,especificidade de 90,47% e uma variabilidade interobservador com taxa de concordância de aproximadamente 30% para os grupos ACB e ASC. A expressão da proteína RAP1 foi positiva na maioria das amostras do grupo “ACB” (15/27 ou55,56%) e resultado negativo na maioria das amostras do grupo “ASC” (4/7 ou 57,14%) com sensibilidade de 16,66%, especificidade de 75, %. As reações imunocitoquímicas das proteínas p16 e Ki-67 demonstraram, em ambos os grupos,apresentaram predomínio absoluto ou totalidade de resultados negativos. O DNA de HPV foi detectado em 9 (33,33%) das 27 amostras do grupo ACB e em 4 (57,14%)das 7 amostras do grupo ASC. O HPV-16 foi detectado nas 4 amostras do grupoASC. Nas amostras do grupo ACB foram detectados os HPV-16, em 5 amostras,HPV-58, em 2 amostras, HPV-45, em 1 amostra e HPV-66 em 1 amostra.Observamos inexistência de relação entre a presença do HPV e a expressão imunocitoquímica de RAP1 em ambos os grupos. Em conclusão, os blocos de células podem complementar o teste de Papanicolaou na triagem do câncer do colo uterino e a expressão da proteína RAP1 está aumentada em células cervicais em ambiente inflamatório, associado ou não à presença de HPV.

Caracterização imunocitoquímica da expressão da proteína RAP1 em blocos de células escamosas provenientes de citologia cervical em meio líquido / Immunocytochemistry characterization of expression of RAP1 protein in squamous cell blocks from cervical cytology in liquid medium

O diagnóstico precoce acurado do câncer cervical, relevante problema de saúde pública no mundo e no Brasil, pela citologia oncótica (Teste de Papanicolaou), é muito prejudicado pela subjetividade e variabilidade dos resultados falsos negativos e falsos positivos do método, particularmente diante de células escamosas atípicas(ASC). Recentes inovações técnicas, como citologia em meio líquido e imunocitoquímica com biomarcadores de proliferação celular, aumentaram a expectativa de melhorias no rastreamento do câncer cervical. No entanto, a aplicabilidade destas inovações nos estágios mais iniciais de atipia e displasias epiteliais permanecem incertas. Assim, considerando resultado prévio do nosso grupo de pesquisa, que identificou a proteína RAP1 como biomarcador diagnóstico da displasia cervical, este trabalho tem como objetivo caracterizar a expressão da proteína RAP1, comparativamente à expressão dos biomarcadores p16 e Ki-67, por imunocitoquímica, em blocos de células escamosas cervicais para possível aplicabilidade na triagem do câncer do colo do útero. Para tal, 34 amostras, 27 pacientes com diagnóstico de alterações celulares benignas (ACB) e 7 pacientes com diagnóstico de ASC foram coletadas na unidade Jenny Faria do Hospital das Clínicas da UFMG. Os resultados indicaram que 85% das amostras de blocos celulares foram satisfatórias para análise morfológica e a técnica reproduziu os principais parâmetros citopatológicos da citologia convencional...

Em relação a sua utilização para diagnóstico, o bloco de células apresentou sensibilidade de 38,46%,especificidade de 90,47% e uma variabilidade interobservador com taxa de concordância de aproximadamente 30% para os grupos ACB e ASC. A expressão da proteína RAP1 foi positiva na maioria das amostras do grupo “ACB” (15/27 ou55,56%) e resultado negativo na maioria das amostras do grupo “ASC” (4/7 ou 57,14%) com sensibilidade de 16,66%, especificidade de 75, %. As reações imunocitoquímicas das proteínas p16 e Ki-67 demonstraram, em ambos os grupos,apresentaram predomínio absoluto ou totalidade de resultados negativos. O DNA de HPV foi detectado em 9 (33,33%) das 27 amostras do grupo ACB e em 4 (57,14%)das 7 amostras do grupo ASC. O HPV-16 foi detectado nas 4 amostras do grupoASC. Nas amostras do grupo ACB foram detectados os HPV-16, em 5 amostras,HPV-58, em 2 amostras, HPV-45, em 1 amostra e HPV-66 em 1 amostra.Observamos inexistência de relação entre a presença do HPV e a expressão imunocitoquímica de RAP1 em ambos os grupos. Em conclusão, os blocos de células podem complementar o teste de Papanicolaou na triagem do câncer do colo uterino e a expressão da proteína RAP1 está aumentada em células cervicais em ambiente inflamatório, associado ou não à presença de HPV...

Mutation analysis of RAP1 gene in papillary thyroid carcinomas.

In human papillary thyroid carcinomas (PTCs), the genetic alterations of RET/PTC, RAS or BRAF account for about 60-70% of cases with practically no overlap, providing strong genetic evidence that constitutive active signaling along MAPK pathway is critical for PTC development. In the remaining 30-40% of the cases, the oncogenes are still unknown. RAP1 is a member of the RAS family of small G proteins transmitting signals from the TSH-R to MAPK pathway using cAMP-dependent mechanism in thyroid cells. RAP1 was shown to have both mitogenic and tumorigenic properties in certain systems; however, the potential role of RAP1 mutation in thyroid carcinogenesis has yet to be elucidated. In this study, we analyzed the mutational status of RAP1 gene in 36 Russian patients with PTCs without RET/PTC rearrangement, BRAF mutation or RAS mutation. No mutations in either RAP1A or RAP1B genes were found. These results suggest that RAP1 mutation does not play an important role in PTC pathogenesis.

The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages.

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.

Effects of prostaglandin E2 on the subcellular localization of Epac-1 and Rap1 proteins during Fcgamma-receptor-mediated phagocytosis in alveolar macrophages.

Recent studies have demonstrated a central role for the exchange protein activated by cAMP (Epac) in the inhibition of Fcgamma-receptor-mediated phagocytosis and bacterial killing by prostaglandin E(2) (PGE(2)) in macrophages. However, the subcellular localization of Epac, and its primary target Rap1, has yet to be determined in primary macrophages. Therefore, we used immunofluorescent techniques and phagosome isolation to localize Epac-1 and Rap1 in alveolar macrophages. Epac-1 was predominantly expressed on punctate and tubular membranes throughout the cell body; on the plasma membrane; and co-localized with microtubule organizing centers (MTOCs). Rap1 was abundant on punctate membranes, less abundant on plasma membrane, and also found on MTOCs. Following PGE(2) treatment, Epac-1, but not Rap1, accumulated on the nuclear envelope and disappeared from MTOCs. By immunofluorescent microscopy, both Epac-1 and Rap1 were seen to associate with phagosomes containing IgG-opsonized beads, but this association appeared weak, as we failed to observe such interactions in phagosomes isolated from cells at various time points after bead ingestion. Strikingly, however, Epac-1, but not Rap1, appeared to accumulate on maturing phagosomes, but only after PGE(2) treatment (or treatment with a selective Epac-1 agonist). This association was confirmed in isolated phagosome preparations. The changes in Epac-1 localization were too slow to account for the inhibitory effects of PGE(2) on phagocytosis. However, the appearance of Epac-1 on late phagosomes following PGE(2) treatment might be important for suppressing H(2)O(2) production and inhibiting the killing of intraphagosomal pathogens. The absence of Rap1 on late phagosomes suggests that the effect of Epac-1 might not require Rap1.

Inverse Rap1 and phospho-ERK expression discriminate the maintenance phase of tolerance and priming of antigen-specific CD4+ T cells in vitro and in vivo.

T cell recognition of Ag can result in priming or tolerance depending on the context in which Ag is recognized. Previously, we have reported that these distinct functional outcomes are associated with marked differences in the amplitude, kinetics, and cellular localization of activated, pERK signals at the level of individual Ag-specific T cells in vitro. Here, we show that the GTPase Rap1, which can antagonize the generation of such pERK signals and has been reported to accumulate in tolerant cells, exhibits an inverse pattern of expression to pERK in individual Ag-specific primed and tolerized T cells. Although pERK is expressed by more primed than tolerized T cells when rechallenged with Ag in vitro, Rap1 is expressed by higher percentages of tolerant compared with primed Ag-specific T cells. Moreover, whereas pERK localizes to the TCR and lipid rafts in primed cells, but exhibits a diffuse cellular distribution in tolerized cells, Rap1 colocalizes with the TCR and lipid raft structures under conditions of tolerance, but not priming, in vitro. This inverse relationship between Rap1 and pERK expression is physiologically relevant, given that we observed the same patterns in Ag-specific T cells in situ, following induction of priming and tolerance in vivo. Together, these data suggest that the maintenance of tolerance of individual Ag-specific T cells may reflect the recruitment of up-regulated Rap1 to the immune synapse, potentially resulting in sequestration of Raf-1 and uncoupling of the TCR from the Ras-ERK-MAPK cascade.

Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP.

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2(ko/ko)). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.

Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion.

RAPL, a protein that binds the small GTPase Rap1, is required for efficient immune cell trafficking. Here we have identified the kinase Mst1 as a critical effector of RAPL. RAPL regulated the localization and kinase activity of Mst1. 'Knockdown' of the gene encoding Mst1 demonstrated its requirement for the induction of both a polarized morphology and integrin LFA-1 clustering and adhesion triggered by chemokines and T cell receptor ligation. RAPL and Mst1 localized to vesicular compartments and dynamically translocated with LFA-1 to the leading edge upon Rap1 activation, suggesting a regulatory function for the RAPL-Mst1 complex in intracellular transport of LFA-1. Our study demonstrates a previously unknown function for Mst1 of relaying the Rap1-RAPL signal to induce cell polarity and adhesion of lymphocytes.

Differential and brain region-specific regulation of Rap-1 and Epac in depressed suicide victims.

CONTEXT: Depression is a major public health problem. Despite many years of research, the molecular mechanisms associated with depression remain unclear. Rap-1, activated in response to many extracellular stimuli, is one of the major substrates of protein kinase A, which participates in myriad physiologic functions in the brain, including cell survival and synaptic plasticity. Rap-1 is also activated directly by cyclic adenosine monophosphate through Epac, and thus participates in mediating physiologic functions independent of protein kinase A. OBJECTIVE: To examine whether the pathogenesis of depression is associated with altered activation and expression of Rap-1, as well as expression of Epac, in depressed suicide victims. DESIGN: Postmortem study. SETTING: Tissues were obtained from the Lenhossek Human Brain Program, Semmelweis University, Budapest, Hungary, and the Brain Collection Program of the Maryland Psychiatric Research Center, Baltimore. PARTICIPANTS: Postmortem brains of 28 depressed suicide victims and 28 nonpsychiatric control subjects. INTERVENTION: Examination of brain tissues. MAIN OUTCOME MEASURES: Rap-1 activation as well as messenger RNA and protein levels of Rap-1 and Epac in prefrontal cortex, hippocampus, and cerebellum. RESULTS: Rap-1 activation was significantly reduced (P<.001) in prefrontal cortex and hippocampus in the suicide group. This was associated with significant reductions in Rap-1 messenger RNA and protein levels (P<.001). In contrast, protein level of only Epac-2 (P<.001) but not Epac-1 (P = .89) was significantly increased in prefrontal cortex and hippocampus of these subjects. These changes were present whether the 2 cohorts were analyzed together or separately. None of the measures showed any significant change in cerebellum in the suicide group. CONCLUSION: Given the importance of Rap-1 in neuroprotection and synaptic plasticity, our findings of differential regulation of Rap-1 and Epac between brain regions suggest the relevance of these molecules in the pathophysiology of depression.

Enhanced induction of prostatic dysplasia and carcinoma in Noble rat model by combination of neonatal estrogen exposure and hormonal treatments at adulthood.

Estrogens have been implicated to play certain but yet undefined roles in the normal and neoplastic growth of prostate gland. Studies of perinatal exposure in rodents demonstrate that effects of perinatal estrogenization are permanent and carcinogenic in prostate gland. In the Noble (Nb) rat model, prostatic dysplasia and neoplastic lesions can be induced by a chronic treatment with both testosterone and estrogen at adulthood. However, by this conventional protocol, neoplastic lesions are mostly confined to the lateral (LP) and ventral (VP) prostates, while gross prostatic tumors are rarely induced. Based on these two experimental models, we developed a modified treatment protocol for the enhancement of prostate cancer induction in Nb rat model by combining neonatal estrogen exposure of male offspring followed by the hormonal treatment at adulthood (NeoE + T-E2). Using this modified protocol, we were able to induce more extensive development of neoplastic lesions in all three prostatic lobes and also gross tumors at relatively high incidence within 6-9 months. Western blottings and immunohistochemistry showed that ERalpha expression was increased in the hypertrophic peri-acinar and -ductal smooth muscle cells while ERbeta and AR expressions are markedly decreased in dysplastic and neoplastic lesions in NeoE + T-E2-treated prostates. Immunohistochemistry showed that expression of three tumor suppressors (BRCA2, PTEN, and Rap1) and tubulin-alpha are markedly decreased in dysplastic and neoplastic lesions. In addition, loss of expression of smooth muscle differentiation markers (desmin, alpha-actin, and vinculin) and defects of basement membranes were also seen in the reactive stroma. These results suggest that exposure to high levels of estrogens, either endogenous or exogenous, in early life could play a role in the development of prostate cancer in later life.

Multicompartmental distribution of the tuberous sclerosis gene products, hamartin and tuberin.

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.

cAMP signaling pathway in depressed patients with psychotic features.

Abnormalities in protein kinase A (PKA) and Rap1 have recently been reported in depressed patients. The aim of the present study was to investigate the levels of these proteins in platelets from untreated unipolar and bipolar depressed patients with psychotic features. The levels PKA and Rap1 were assessed by Western blot analysis and immunostaining in 37 drug-free patients and 29 healthy subjects. Both unipolar and bipolar patients with psychotic depression have significantly lower levels of platelet regulatory type I and higher levels of catalytic subunits of PKA than controls, whereas the levels of regulatory type II were higher only in psychotic unipolar patients. No significant differences were found in the immunolabeling of both Rap1 and actin among groups. These findings support the idea that besides nonpsychotic depression, abnormalities of PKA could be linked, albeit in a somewhat different way, with psychotic depression.

Protein kinase A and Rap1 levels in platelets of untreated patients with major depression.

We have recently reported altered levels of protein kinase A and Rap1 in patients with bipolar disorder. The purpose of the current investigation was to assess the levels of these proteins in platelets from untreated euthymic and depressed patients with major unipolar depression. Platelets were collected from 45 drug-free unipolar patients (13 euthymic and 32 depressed) and 45 healthy subjects. The levels of protein kinase A and Rap1 were assessed by Western blot analysis, immunostaining and computer-assisted imaging. The immunolabeling of the regulatory subunit type II of protein kinase A and that of Rap1 was significantly lower in untreated depressed patients compared with untreated euthymic patients and healthy subjects. No significant differences were found in the immunolabeling of both the regulatory type I and the catalytic subunits of protein kinase A among groups. Levels of the regulatory subunit type II of protein kinase A and Rap1 are altered in platelets of unipolar depressive patients. These findings may provide new insight about the relationship between components of cAMP signaling and affective disorders.

Localization of a low Mr GTP-binding protein, rap1 protein, in plasma membranes and secretory granule membranes of rat parotid gland.

Subcellular fractions were prepared from rat parotid gland by sequential centrifugation, Percoll gradient centrifugation and divalent-cation precipitation, and the localization of a low Mr GTP-binding protein, rap1 protein (rap1p) was analyzed by immunoblotting using a specific antibody. rap1p was found to be located in apical and basolateral plasma membranes, and secretory granule membranes in rat parotid gland. On the other hand, the beta gamma subunits of heterotrimeric Gt protein was localized in plasma membranes but not in granule membranes.