Abstract: OBJECTIVE: The primary aim of this study was to investigate the role of angiogenesis and inflammatory cell response in cervical carcinogenesis. METHODS: Formalin-fixed tissue specimens from 58 uterine cervical specimens (8 CIN1, 14 CIN2, 28 CIN3, and 8 SCC), representing the different stages of cervical carcinogenesis, were immunohistochemically analyzed. Normal cervical tissue specimens were also included as controls. The present study assessed the expression of CD31 and CD105 to evaluate microvessel density (MVD), the macrophage marker CD68 and the panleukocyte marker CD45. In addition, expression of iNOS (inducible Nitric Oxide Synthase) was also evaluated. RESULTS: MVD, measured by either CD31 or CD105, increased along the continuum from normal epithelium to squamous cell carcinoma, and a significant correlation between the CD105-MVD and the CD31-MVD was observed (r=0.8735; p<0.0001). Furthermore, the number of infiltrating macrophages was significantly associated with progression to malignancy. Interestingly, there was a close positive correlation between macrophage counts and CD105-MVD (r=0.7525; p<0.0001). In striking contrast to the other angiogenic and inflammatory markers tested, iNOS expression was significantly reduced as cervical lesion grade progressed from low to high. CONCLUSIONS: Our findings demonstrated a positive correlation between neovascularity and macrophage counts, whereas iNOS expression displayed an inverse relationship with macrophage density and tumor progression. Low iNOS expression may modify the function of tumor-infiltrating macrophages toward a malignant phenotype that promotes tumor progression rather than an anti-tumor response.
Abstract: Keratinocytes, the target cell of human papillomavirus (HPV) infection, can produce numerous cytokines and pro-inflammatory molecules which are important for the generation of an effective immune response. How this biological response, which involves the tumor stroma, is affected by the HPV oncoproteins within the epithelial cell itself is not clear. Here it is shown that oncoproteins of different HPV genotypes (alpha- versus beta-HPV genus) alter the expression of pro-inflammatory molecules in early passage primary human keratinocytes and the immortalized cell line HaCaT. HPV5 E6/E7 oncoproteins significantly induced interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) expression. By contrast, the same molecules were down-regulated or not modulated in HPV16 E6/E7 transduced keratinocytes. Interestingly, HPV38 oncoproteins expression resulted in a lower induction of pro-inflammatory molecules, resembling the behavior displayed by the mucosal carcinogenic HPV16. Finally, inducible nitric oxide synthase (iNOS) expression levels and nitric oxide (NO) production were induced at similar levels by all the HPV genotypes tested. These results further emphasize the different biological activities among HPV genotypes, and offer new insights into HPV-associated skin diseases.
Abstract: BACKGROUND: In a previous analysis of head and neck squamous cell carcinomas (HNSCCs), we showed that the levels of the interferon-inducible protein IFI16 inversely correlate with cancer grade. In this study, we further evaluate the molecular role of IFI16 in the development of HNSCCs. METHODS: The effect of IFI16 expression was evaluated by its retroviral restoration in an IFI16-negative HNSCC-derived cell line, HNO136. Growth rate and soft agar colony formation were evaluated. The effect of IFI16 restoration in cells exposed to doxorubicin was also analyzed. RESULTS: IFI16 restoration resulted in the inhibition of both cell growth and in vitro transforming activity and increased doxorubicin-induced cell death by accumulating the cells at the G2/M phase. CONCLUSION: In agreement with our previous in vivo data, IFI16 appears to be involved in maintaining the normal growth of epithelial cells, whereas its downregulation may contribute to uncontrolled cell proliferation and tumorigenesis.
Abstract: The p53R2 protein is a p53-inducible small subunit of ribonucleotide reductase. It plays a crucial role in p53-dependent cellular response to DNA damage and oxidative stress by providing deoxyribonucleotides (dNTPs) to the DNA repair machinery and by scavenging reactive oxygen species (ROS). To investigate the effects of high-risk human papillomavirus (HPV) oncoproteins on p53R2 expression after DNA damage, we analyzed the p53R2 protein levels in human cells ectopically expressing the HPV-16 E6 and E7 genes, and in the HPV-positive cancer cell lines SiHa, CaSki and HeLa, exposed to adriamycin or to H(2)O(2). We found that in normal cells, p53R2 expression is efficiently induced by both H(2)O(2) and adriamycin, supporting the role of p53R2 in cellular response to oxidative stress. Ectopic expression of E6 impaired p53 and p53R2 induction after DNA damage in human fibroblasts. Moreover, SiHa, CaSki and HeLa cells were unresponsive to H(2)O(2) exposure, and adriamycin induced p53R2 levels only in SiHa cells. Our results imply that high-risk HPV infection may suppress the p53R2-dependent dNTPs supply to the DNA repair system and the ROS scavenging activity; they also suggest that an altered p53R2 response to genotoxins and to oxidative stress may contribute to HPV-induced genetic instability and carcinogenesis.
Abstract: OBJECTIVE: To investigate the presence and clinical significance of autoantibodies against the interferon-inducible gene IFI16 in systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and other autoimmune diseases. METHODS: Immunohistochemical analysis was used to evaluate the expression of IFI16 in skin biopsy specimens obtained from patients with SSc and patients with SLE. Levels of antibodies against IFI16 in sera from 82 patients with SSc and 100 patients with SLE were determined by enzyme-linked immunosorbent assay. Other autoimmune diseases such as primary Sjögren's syndrome (SS), rheumatoid arthritis (RA), chronic urticaria, and hepatitis C virus (HCV) infection were also examined. RESULTS: Expression of IFI16 was greatly increased and was ubiquitous in all layers of the epidermis and in the dermal inflammatory infiltrates of lesional skin from both patients with SLE and patients with SSc. Patients with SLE, those with primary SS, and those with SSc exhibited significantly higher anti-IFI16 IgG antibody levels compared with normal controls (for SLE, P < 0.002; for primary SS, P < 0.001; for SSc, P < 0.0005). Anti-IFI16 titers above the ninety-fifth percentile for control subjects were observed in 26% of the patients with SLE, 50% of those with primary SS, and 21% of those with SSc (28% of patients with limited cutaneous SSc [lcSSc] versus 4% of patients with diffuse cutaneous SSc [dcSSc]). In contrast, the prevalence of anti-IFI16 was 4% in patients with RA, 5% in those with chronic urticaria, and 13% in those with HCV infection. CONCLUSION: The results of this study provide evidence that an IFN-inducible gene, IFI16, may be involved in the pathophysiologic mechanisms of connective tissue disorders such as SSc. Moreover, a strict correlation with lcSSc was also demonstrated, thus providing a novel tool in the differential diagnosis of lcSSc from dcSSc.
Abstract: AIMS: To investigate whether the expression of interferon (IFN)-inducible gene IFI16 is inversely related to proliferative activity in vivo, we compared immunohistochemical reactivity of IFI16 in a series of head and neck squamous cell carcinomas (HNSCCs) with their proliferation index and the cell cycle regulator pRb. As human papillomavirus (HPV) infection is manifested by changes in the function or expression level of host genes such as IFN-inducible genes, we also investigated the presence of HPV DNA to determine whether head and neck cancers associated with HPV DNA can be distinguished from tumours that are presumably transformed by other mechanisms. METHODS: Thirty-six HNSCCs were evaluated for IFI16, pRb and Ki67 expression by immunohistochemistry. The presence of HPV was also detected by polymerase chain reaction. Nine tumours were located in the oropharynx (tonsillar area) and 27 in the larynx. RESULTS: HPV DNA was found in 14 of 25 (56%) laryngeal SCCs and in five of nine (56%) tonsillar SCC specimens examined; 17 out of the 19 HPV-DNA-positive cases showed high-grade IFI16 expression. Overall, proliferative activity was significantly related to tumour differentiation and histological grading. IFI16 protein expression was significantly inversely correlated with Ki67 (P = 0.039). Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index. CONCLUSIONS: To our knowledge, this is the first expression analysis of the IFN-inducible IFI16 gene in HNSCC. Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index.
Abstract: p204 overexpression in retinoblastoma (Rb)-/- mouse embryo fibroblasts or transfection of p204 mutated at both Rb-binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double-mutant (p204dm) harboring both the C-terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm-expressing cells than vector control cells. The p204dm-transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low-serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb-binding motifs, can become oncogenic.
Abstract: This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN.
Abstract: We have used yeast two-hybrid screening to isolate cDNA-encoding proteins interacting with the protein encoded by the interferon (IFN)-inducible gene Ifi204. Four independent overlapping clones were isolated from an NIH3T3 cDNA library. The largest clone encoded a protein (1203 amino acids in length) sharing 94% identity with the C-terminal portion of the human translocated promoter region (Tpr) protein. Northern blot analysis revealed a 7.5-kilobase mRNA present in both mouse and human cell lines. In addition, in vivo interaction was demonstrated by coimmunoprecipitation experiments. Anti-Tpr polyclonal monospecific antibodies (Ab) used for immunofluorescence staining labeled the nuclear envelope (NE) in a punctate pattern characteristic of nucleoporins and also yielded staining throughout the nuclear interior. The intranuclear Tpr occurred in apparently discrete foci. When superimposed on optical sections obtained with anti-p204 Abs, these colocalized, with the sole exception of the nucleolar compartment stained by the anti-p204 Abs only. Although the specific function of Tpr is not defined, it appears to mediate p204 translocation from the cytoplasmic to the nuclear compartment following IFN treatment.
Abstract: We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.
Abstract: To examine whether Ifi 200 genes are involved in antiviral state induction by IFNs we expressed mutant forms capable of inactivating the endogenous p204 and analyzed replication of both RNA and DNA viruses following IFN-alpha treatment. Inactivation of p204 does not impair replication of vesicular stomatitis virus, encephalomyocarditis virus, ectromelia virus, and herpes simplex virus 1 and does not alter an IFN-alpha induced antiviral state. By contrast, in cells lacking functional p204, mouse cytomegalovirus (MCMV) replication is strongly inhibited and is not further modulated by IFN-alpha. These results suggest that p204, a member of the Ifi 200 gene family, is not involved in the IFN-alpha-induced antiviral activity against some RNA or DNA viruses, but is required by MCMV for its replication.
Abstract: Recent evidence has shown that human papillomavirus (HPV) is involved in both the development of carcinoma and in premalignant mucosal lesions of the oral cavity. This study examined the relationship of HPV infection to some pathological features in precancerous lesions of the larynx, not examined extensively so far. Fifty formalin-fixed paraffin-embedded tissue sections containing human laryngeal precancerous lesions were screened for the presence of HPV infection by polymerase chain reaction, and for capsid protein expression by immunohistochemistry with polyclonal antibody directed against the L1 protein. The presence of HPV DNA was detected in 28 of 50 specimens (56%), including 9/12 cases with mild dysplasia (75%), 3/6 cases with moderate dysplasia (50%), and 7/11 cases with severe dysplasia (64%). Multiple HPV infections, containing two or three types, were detected in 17 of the 28 HPV-positive lesions (60%). Of 21 cases with keratosis and no dysplasia, 11 were positive for HPV DNA (52%) and 4 showed L1 staining (36%). By contrast, L1 positivity was revealed only in two lesions with moderate dysplasia, confirming that fully productive HPV infection is strictly dependent on epithelial differentiation and surface keratinization. The probability that HPV is a cofactor in the malignant progression of these lesions is suggested by the fact that 3/4 patients who developed cancer within 50 months were positive for HPV DNA.
Abstract: Iron may be important in catalyzing excessive production of reactive oxygen species (ROS). Cellular iron homeostasis is regulated by iron regulatory proteins (IRPs), which bind to iron-responsive elements (IRE) of mRNAs for ferritin and transferrin receptor (TfR) modulating iron uptake and sequestration, respectively. Although iron is the main regulator of IRP activity, IRP is also influenced by other factors, including the redox state. Therefore, IRP might be sensitive to pathophysiological alterations of redox state caused by ROS. However, previous studies have produced diverging evidence on the effect of oxidative injury on IRP. Results obtained in an animal model close to a pathophysiological condition, such as ischemia reperfusion of the liver as well as in a cell-free system involving an enzymatic source of O2 and H2O2, indicate that IRP is downregulated by oxidative stress. In fact, IRP activity is inhibited at early times of post-ischemic reperfusion. Moreover, the concerted action of O2 and H2O2 produced by xanthine oxidase in a cell-free system caused a remarkable inhibition of IRP activity. IRP seems a direct target of ROS; in fact, in vivo inhibition can be prevented by the antioxidant N-acetylcysteine and by interleukin-1 receptor antagonist. In addition, modulation of iron levels of the cell-free assay did not affect the downregulation imposed by xanthine oxidase. Conceivably, downregulation of IRP activity by O2 and H2O2 may facilitate iron sequestration into ferritin, thus limiting the pro-oxidant challenge of iron.
