Rb-E2F-HDAC Repressor Complexes Control Interferon-Induced Repression of Adenovirus To Promote Persistent Infection.

Interferons (IFNs) are cytokines that induce a global change in the cell to establish antiviral immunity. We previously demonstrated that human adenovirus (HAdV) exploits IFN-induced viral repression to persist in infected cells. Although this in vitro persistence model has been described, the mechanism behind how persistent HAdV infection is established is not well understood. In this study, we demonstrate that IFN signaling is essential for viral repression and promoting persistent infection. Cyclin-dependent kinase 4 (CDK4), an antagonist of retinoblastoma (Rb) family proteins, was shown to disrupt the viral repression induced by IFNs. Consistent with this result, knockout of the Rb family proteins pRb, p107, and/or p130 drastically reduced the effect of IFNs on viral replication. The pRb protein specifically contributed the greatest effect to IFN inhibition of viral replication. Interestingly, IFNs did not impact pRb through direct changes in protein or phosphorylation levels. Cells treated with IFNs continued to cycle normally, consistent with observations that persistently infected cells remain for long periods of time in the host and in our in vitro persistent infection model. Finally, we observed that histone deacetylase (HDAC) inhibitors activated productive viral replication in persistently infected cells in the presence of IFN. Thus, HDACs, specifically class I HDACs, which are commonly associated with Rb family proteins, play a major role in the maintenance of persistent HAdV infection in vitro. This study uncovers the critical role of pRb and class I HDACs in the IFN-induced formation of a repressor complex that promotes persistent HAdV infections. IMPORTANCE Adenoviruses are ubiquitous viruses infecting more than 90% of the human population. HAdVs cause persistent infections that may lead to serious complications in immunocompromised patients. Therefore, exploring how HAdVs establish persistent infections is critical for understanding viral reactivation in immunosuppressed individuals. The mechanism underlying HAdV persistence has not been fully explored. Here, we provide insight into the contributions of the host cell to IFN-mediated persistent HAdV infection. We found that HAdV-C5 productive infection is inhibited by an Rb-E2F-HDAC repressor complex. Treatment with HDAC inhibitors converted a persistent infection to a lytic infection. Our results suggest that this process involves the noncanonical regulation of Rb-E2F signaling. This study provides insight into a highly prevalent human pathogen, bringing a new level of complexity and understanding to the replicative cycle.

BCG immunotherapy inhibits cancer progression by promoting the M1 macrophage differentiation of THP­1 cells via the Rb/E2F1 pathway in cervical carcinoma.

Bacillus Calmette­Guérin (BCG) immunotherapy increases macrophage polarization toward M1­type macrophages. In the present study, to identify the M1/M2 marker genes in the carcinogenesis and progression of cervical cancer, the microarray datasets GSE9750 and GSE7803 were downloaded from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the University of California Santa Cruz (UCSC) Xena browser. Survival analysis revealed that M1 markers (IL­12) were involved in anti­tumour progression, and M2 markers (IL­10) were involved in the carcinogenesis and invasion of cervical cancer. The expression of M1 markers (IL­12, inducible nitric oxide synthase and CD80) and M2 markers (IL­10 and arginase) was examined to determine whether BCG affects the polarization of macrophages and to elucidate the underlying mechanisms. The results revealed that BCG promoted macrophage polarization towards the M1 phenotype and enhanced the transition of M2 to M1 macrophages. The results also revealed that polarized M1 macrophages induced by BCG decreased the protein expression of phosphorylated (p­)retinoblastoma (Rb)/E2F transcription factor 1 (E2F1), inhibited the proliferation and promoted the apoptosis of HeLa cells. On the whole, these results demonstrated that BCG promoted the anti­tumour progression of M1 macrophages and inhibited the pro­tumour activation of M2 macrophages via the Rb/E2F1 signalling pathway in HeLa cells. This suggests the possibility of a direct translation of this combination strategy to clinical practice for the treatment of cervical cancer.

Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer.

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.

Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression.

Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.

Recruitment of Antigen Presenting Cells to Skin Draining Lymph Node From HPV16E7-Expressing Skin Requires E7-Rb Interaction.

"High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRbΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRbΔL/ΔL skin grafts. Here, we further show that K14.E7xRbΔL/ΔL skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b+ antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRbΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRbΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRbΔL/ΔL keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.

Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps.

Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.

Cutting Edge: Foxp1 Controls Naive CD8+ T Cell Quiescence by Simultaneously Repressing Key Pathways in Cellular Metabolism and Cell Cycle Progression.

Previously we have shown that transcription factor Foxp1 plays an essential role in maintaining naive T cell quiescence; in the absence of Foxp1, mature naive CD8(+) T cells proliferate in direct response to homeostatic cytokine IL-7. In this study, we report that the deletion of Foxp1 in naive CD8(+) T cells leads to enhanced activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway and its downstream cell growth and metabolism targets in response to IL-7. We found that Foxp1 directly regulates PI3K interacting protein 1, a negative regulator of PI3K. Additionally, we found that deletion of Foxp1 in naive CD8(+) T cells results in increased expression levels of E2fs, the critical components for cell cycle progression and proliferation, in a manner that is not associated with increased phosphorylation of retinoblastoma protein. Taken together, our studies suggest that Foxp1 enforces naive CD8(+) T cell quiescence by simultaneously repressing key pathways in both cellular metabolism and cell cycle progression.

E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: cancer cell expression of HLA-DR.

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA-DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.

Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein.

OspF, OspG and IpaH(9.8) are type III secretion system (T3SS) effectors of Shigella flexneri that downregulate the host innate immune response. OspF modifies mitogen-activated protein kinase pathways and polymorphonuclear leucocyte transepithelial migration associated with Shigella invasion. OspF also localizes in the nucleus to mediate chromatin remodelling, resulting in reduced transcription of inflammatory cytokines. We now report that OspB can be added to the set of S. flexneri T3SS effectors required to modulate the innate immune response. T84 cells infected with a Delta ospB mutant resulted in reduced polymorphonuclear leucocyte transepithelial migration and mitogen-activated protein kinase signalling. Tagged versions of OspB localized with endosomes and the nucleus. Further, T84 cells infected with the Delta ospB mutant showed increased levels of secreted IL-8 compared with wild-type infected cells. Both GST-OspB and GST-OspF coprecipitated retinoblastoma protein from host cell lysates. Because Delta ospB and Delta ospF mutants share similar phenotypes, and OspB and OspF share a host binding partner, we propose that OspB and OspF facilitate the remodelling of chromatin via interactions with retinoblastoma protein, resulting in diminished inflammatory cytokine production. The requirement of multiple T3SS effectors to modulate the innate immune response correlates to the complexity of the human immune system.

Disruption of mutually negative regulatory feedback loop between interferon-inducible p202 protein and the E2F family of transcription factors in lupus-prone mice.

Studies have identified IFN-inducible Ifi202 gene as a lupus susceptibility gene (encoding p202 protein) in mouse models of lupus disease. However, signaling pathways that regulate the Ifi202 expression in cells remain to be elucidated. We found that steady-state levels of Ifi202 mRNA and protein were high in mouse embryonic fibroblasts (MEFs) from E2F1 knockout (E2F1(-/-)) and E2F1 and E2F2 double knockout (E2F1(-/-)E2F2(-/-)) mice than isogenic wild-type MEFs. Moreover, overexpression of E2F1 in mouse fibroblasts decreased expression of p202. Furthermore, expression of E2F1, but not E2F4, transcription factor in mouse fibroblasts repressed the activity of 202-luc-reporter in promoter-reporter assays. Interestingly, the E2F1-mediated transcriptional repression of the 202-luc-reporter was independent of p53 and pRb expression. However, the repression was dependent on the ability of E2F1 to bind DNA. We have identified a potential E2F DNA-binding site in the 5'-regulatory region of the Ifi202 gene, and mutations in this E2F DNA-binding site reduced the E2F1-mediated transcriptional repression of 202-luc-reporter. Because p202 inhibits the E2F1-mediated transcriptional activation of genes, we compared the expression of E2F1 and its target genes in splenic cells from lupus-prone B6.Nba2 congenic mice, which express increased levels of p202, with age-matched C57BL/6 mice. We found that increased expression of Ifi202 in the congenic mice was associated with inhibition of E2F1-mediated transcription and decreased expression of E2F1 and its target genes that encode proapoptotic proteins. Our observations support the idea that increased Ifi202 expression in certain strains of mice contributes to lupus susceptibility in part by inhibiting E2F1-mediated functions.

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods.

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.

SPR-based immunocapture approach to creating an interfacial sensing architecture: Mapping of the MRS18-2 binding site on retinoblastoma protein.

Biosensor technologies based on optical readout are widely used in protein-protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb-MRS18-2 protein complex was examined and the pRb binding site (A-box-spacer-B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein-Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.

Levalbuterol inhibits human airway smooth muscle cell proliferation: therapeutic implications in the management of asthma.

BACKGROUND: Racemic albuterol is a mixture of (R)- and (S)-enantiomers of albuterol. Its pharmacological activity and clinical efficacy reside in the (R)-enantiomer (levalbuterol), but the (S)-enantiomer exacerbates airway reactivity in nonclinical models. The role of albuterols in airway smooth muscle cell (SMC) proliferation is not well understood. METHODS: The effect of levalbuterol on human bronchial SMC growth was compared with the effects of racemic albuterol and (S)-albuterol. Cells were fed albuterols and 3H-thymidine in 5% FBS and incubated for 24 h. The effect of (S)-albuterol on levalbuterol actions was also studied and so were the effects of cAMP/PKA, PI-3 kinase, NK-kappaB, and retinoblastoma (Rb) proteins on albuterols and human bronchial SMC proliferation. RESULTS: Levalbuterol inhibited cell proliferation at low concentrations. The growth-inhibitory effect of levalbuterol occurs via activation of the cAMP/PKA pathway. Addition of (S)-albuterol to levalbuterol decreased the growth-inhibitory effect of levalbuterol, and (S)-albuterol attenuated levalbuterol-induced cAMP release by 65%. Levalbuterol inhibited NF-kappaB and Rb protein expressions. ICI-118551 abrogated the inhibitory properties of levalbuterol. The PAF receptor antagonist CV-3988 inhibited (S)-albuterol-induced cell growth, with no effect on levalbuterol. CONCLUSIONS: Levalbuterol inhibits cell growth by activating the cAMP/PKA pathway and inhibiting PI-3 kinase, NF-kappaB and Rb protein expression, and (S)-albuterol induces cell growth by activating PAF-receptor-mediated cell signaling.

Role of cdk4, p16INK4, and Rb expression in the prognosis of bronchioloalveolar carcinomas.

BACKGROUND: The p16(INK4) protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. OBJECTIVES: Loss of p16(INK4) has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. METHODS: We examined immunohistochemical expression of p16(INK4), cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. RESULTS: All BACs expressed cdk4, while 89 and 82% expressed p16(INK4) and Rb proteins, respectively. None of the clinicopathological factors correlated with p16(INK4), cdk4, or Rb expression separately. A low p16(INK4)/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16(INK4)/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16(INK4)/cdk4 ratio (p = 0.01). CONCLUSIONS: The results suggest a role of the cdk4/p16(INK4) pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16(INK4)/cdk4 ratio may identify tumors that are destined to behave unfavorably.

The proliferation inhibitory proteins p27(Kip1) and retinoblastoma are involved in the control of equine lymphocyte proliferation.

Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.

Prognostic relevance of altered pRb and p53 protein expression in surgically treated non-small cell lung cancer patients.

OBJECTIVE: The prognostic value of pRb and p53 altered expression in non-small cell lung cancer (NSCLC) remains debatable. We assessed the occurrence of altered pRb and p53 protein expression, and the prognostic value of these assays considered as separate and combined variables in operable NSCLC. The study group included 195 NSCLC consecutive patients from one institution who underwent curative pulmonary resection between 1994 and 1999. METHODS: Expression of pRb and p53 was assessed immunohistochemically with the use of monoclonal antibodies (LM95.1 and Pab 1801, Oncogene Science, respectively). RESULTS: A lack of pRb and abnormal p53 protein expression were found in 57 (29%) and 92 samples (47%), respectively, whereas both abnormalities (pRb-/p53+) occurred in 24 samples (12%). There was no relationship between altered pRb/p53 expression and major clinico-pathological characteristics, neither was there a significant difference in disease-free and overall survival between particular groups of patients with tumors carrying four possible pRb/p53 phenotypes. In uni- and multivariate analysis, the only variable associated with shortened disease-free and overall survival was stage of disease (p < 0.001) and degree of tumor differentiation (p = 0.005). CONCLUSION: These results suggest that altered pRb and p53 expression does not provide prognostic information in operable NSCLC patients.

Expression of p27(kip1), Rb protein and proliferating cell nuclear antigen and its relationship with clinicopathology in human pancreatic cancer.

OBJECTIVE: To investigate the effect of inhibiting factor of cell cycle regulation p27(kip1), retinoblastinoma protein (Rb protein), and proliferating cell nuclear antigen (PCNA) on the genesis and progression of human pancreatic cancer. METHODS: The expression of p27(kip1), Rb protein and PCNA in the tumor tissue and adjacent tissue of 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS: The p27(kip1) protein positive-expression rate in the tumor tissue of pancreatic cancer was 56.25%, which was lower than that in the adjacent pancreatic tissue (P<0.05). p27(kip1) protein positive-expression was correlated significantly with tumor cell differentiation and lymph node metastasis (P<0.05). The Rb gene protein positive-expression rate in the tumor tissue was 50%, which was also lower than that in the adjacent pancreatic tissue (P<0.05). The PCNA positive-expression rate was 71.87%, which was higher than that in the adjacent pancreatic tissue (P<0.05). PCNA positive-expression was also correlated significantly with tumor cell differentiation and lymph node metastasis (P<0.05). CONCLUSION: The decreased expression of p27(kip1), Rb protein and over-expression of PCNA may play an important role in the genesis and progression of pancreatic cancer.

Growth factors and receptors in juvenile nasopharyngeal angiofibroma and nasal polyps: an immunohistochemical study.

BACKGROUND: Juvenile nasopharyngeal angiofibroma is a rare nasopharyngeal tumor that occurs exclusively in adolescent boys. It is a histologically benign but locally persistent growth of stromal and vascular tissue. Although male hormones and some growth factors, such as transforming growth factor beta1 (TGF-beta1), insulin-like growth factor II (IGF-II), and, lately, the proto-oncogene beta-catenin, have been implicated in the histogenesis of the tumor, the biologic signaling pathways that drive this peculiar fibrovascular proliferation are still nuclear. OBJECTIVE: To evaluate immunoexpressions of beta-catenin, c-Kit, p130Cas, TGF-beta3, bone morphogenic protein 4, nerve growth factor (NGF), and the IGF receptor (IGF-1R) in a series of juvenile nasopharyngeal angiofibromas and to compare to that of a group of nasal polyps. DESIGN: A standard immunohistochemical technique was used on paraffin sections of 12 sporadic juvenile nasopharyngeal angiofibromas and 15 nasal polyps with microwave or steam antigen retrieval. Immunoreactivity was analyzed semiquantitatively in stromal cells and endothelial cells of each case. RESULTS: The expressions of beta-catenin (nuclear), c-Kit (cytoplasmic), and NGF (cytoplasmic) were higher and more frequent in stromal cells of juvenile nasopharyngeal angiofibromas than those of nasal polyps. Both juvenile nasopharyngeal angiofibromas and nasal polyps showed similarly frequent and strong immunoreactivity for p130Cas and TGF-beta3 and weak immunoreactivity for bone morphogenic protein 4 in both stromal cells and endothelial cells. No IGF-1R immunoreactivity was detected in any case of either group. CONCLUSIONS: Our results support the role of beta-catenin in juvenile nasopharyngeal angiofibromas and suggest a potential involvement of c-Kit and NGF signaling pathways in the juvenile nasopharyngeal angiofibromas. Although the biologic significance of c-Kit in juvenile nasopharyngeal angiofibromas has yet to be defined, the finding of frequent and high c-Kit expression might have therapeutic importance for patients with juvenile nasopharyngeal angiofibromas.