Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.
SPECIFIC AIMSWe have devised an in vivo experimental model to investigate the role of the human papilloma virus (HPV) type 16 E6 and E7 oncoproteins in the development of radiation resistance in advanced cervical carcinoma. HPV negative human C33A cervical carcinoma cells were transfected with E6 and E7 cDNAs and the transfectants subcutaneously (s.c.) transplanted into scid mice. Tumor growth and radiation resistance under normal and hypoxic conditions were assessed in male and female recipients. Patterns of gene expression were investigated in radiation-resistant vs. radiation-sensitive tumors.PRINCIPAL FINDINGS1. Constitutive expression of the E6 protein promotes rapid tumor developmentE6-expressing tumors reproducibly developed more quickly than E7 tumors, which in turn developed more quickly than vector-only control tumors (Fig. 1a⤻ ), although no difference in growth rate was observed in vitro between E6, E7, or control cell lines. Once established, however, tumor growth rates were similar. There wa...
CD105 (endoglin), a receptor for transforming growth factor beta (TGFbeta), is highly expressed in tissue-cultured, activated endothelial cells in vitro and in tissues undergoing angiogenesis in vivo. The absence of CD105 in knockout mice leads to their death from defective vascular development, but the role of CD105 in the modulation of angiogenesis has not been elucidated. TGFbeta1 is a well-recognized regulator of angiogenesis. Using an antisense approach, we have shown that inhibition of CD105 protein translation in cultured human endothelial cells enhances the ability of TGFbeta1 to suppress growth and migration in these cells. The ability of endothelial cells to form capillary tubes was evaluated by the use of a 3-dimensional collagen matrix system where TGFbeta1 not only reduced the length of capillary-like structures, but also caused massive mortality in CD105-deficient cells compared to control cultures. These results provide direct evidence that CD105 antagonizes the inhibitory effects of TGFbeta1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.
Abstract Haematologists are increasingly using molecular biology methods to detect changes in the RNA levels of specific marker genes. Such changes reflect both the proliferative state and the developmental stage of a haemopoietic cell. RNA analysis by northern hybridization can be used to observe gross changes of gene expression, whereas in situ mRNA hybridization coupled with morphology, identifies single cells expressing a specific gene within complex populations of cell types. Both types of analysis can be used for assessing the expression of genes introduced into haemopoietic cells using the methods described in Chapter 10. In this chapter, we give protocols for the isolation of total cellular RNA, northern hybridization, and in situ RNA hybridization adapted for use with haemopoietic cells. The reader is referred to other molecular biology manuals for further descriptions of these methods (1-4) and other methods of analyzing gene expression including nuclease Sl/RNase protection assays (4), cDNA cloning (4), and polymerase chain reaction analysis of cDNA (5).
Worldwide, at least 170 million people are infected with hepatitis C virus (HCV), which is associated with hepatocellular carcinoma (HCC). With the recent success of Sofosbuvir (and other agents) antiviral therapy may be used as a future early-stage HCC treatment; however, in the short term, a cost-effective solution is needed to treat patients with viral-associated HCC. Here, we emphasize the potential of targeting gap junction intercellular communication (GJIC) as a therapeutic approach for HCC as HCV perturbs GJIC, which is linked to cellular transformation. We review the ROCK inhibitor Y-27632 and structurally related compounds that may inhibit the carcinogenic properties of HCV.
Background: Characterisation of human papilloma virus (HPV) infection in anal squamous cell carcinoma (ASCC) may have dual importance: first, aetiological; second, prognostic, informing outcome after chemo-radiotherapy (CRT). We undertook HPV genotyping, and allelic characterisations, to evaluate the aetiological role of HPV while simultaneously evaluating the impact of HPV genotyping on relapse-free (RFS) and overall survival (OS).
Method: Dual-primer HPV genotyping (subtypes 6, 11, 16, 18, 31, 33, 45, 52, 58) and DNA sequencing of HPV 16 positive tumours were analyzed in 151 consecutively referred ASCCs, previously characterized by immunohistochemistry for p16 expression. In 110 patients treated with CRT, factors influencing RFS and OS were evaluated using univariate and multivariate models.
Results: HPV positivity was observed in 95%. HPV 16 accounted for 89%; of these, 64% harboured the T350G E6 variant. HPV 16 positivity was significantly correlated with improved 5-year RFS (62% v 40%; p = 0.027) and OS (59% v 38%; p = 0.019). p16 expression was also significantly correlated with improved 5-year RFS (65% v 16%; p < 0.0001) and OS (63% v 13%; p < 0.0001). In multivariable models that included HPV 16 status, p16 status, sex, and age, p16 expression remained an independent prognostic factor for RFS (p < 0.0001) and OS (p= 0.002).
Conclusion: In ASCC, near-universal HPV detection rates were demonstrated, higher than generally reported in the literature, and supporting the development of multivalent HPV vaccinations for prevention. By contrast, p16 expression, but not HPV 16 genotype, is an independent prognosticator after chemo-radiotherapy in patients with ASCC.
Abstract Head and neck squamous cell carcinoma (HNSCC) is a widely prevalent cancer globally with high mortality and morbidity. We report here changes in the genomic landscape in the development of HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Frequent likely pathological mutations are restricted to a relatively small set of genes including TP53, CDKN2A , FBXW7 , FAT1 , NOTCH1 and KMT2D ; these arise early in tumour progression and are present in PPOLs with NOTCH1 mutations restricted to cell lines from lesions that subsequently progressed to HNSCC. The most frequent genetic changes are of consistent somatic copy number alterations (SCNA). The earliest SCNAs involved deletions of CSMD1 (8p23.2), FHIT (3p14.2) and CDKN2A (9p21.3) together with gains of chromosome 20. CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines (promoter hypermethylation ~63%, hemizygous deletions ~75%, homozygous deletions ~18%). Forced expression of CSMD1 in the HNSCC cell line H103 showed significant suppression of proliferation (p=0.0053) and invasion in vitro (p=5.98X10 −5 ) supporting a role for CSMD1 inactivation in early head and neck carcinogenesis. In addition, knockdown of CSMD1 in the CSMD1 -expressing BICR16 cell line showed significant stimulation of invasion in vitro (p=1.82 x 10 −5 ) but not cell proliferation (p=0.239). HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1 , hsa-miR-548k and TP63 in the metastases group. GISTIC peak SCNA regions showed significant enrichment (adj P<0.01) of genes in multiple KEGG cancer pathways at all stages with disruption of an increasing number of these involved in the progression to lymph node metastases. Sixty-seven genes from regions with statistically significant differences in SCNA/LOH frequency between immortal PPOL and HNSCC cell lines showed correlation with expression including 5 known cancer drivers. Lay Summary Cancers affecting the head and neck region are relatively common. A large percentage of these are of one particular type; these are generally detected late and are associated with poor prognosis. Early detection and treatment dramatically improve survival and reduces the damage associated with the cancer and its treatment. Cancers arise and progress because of changes in the genetic material of the cells. This study focused on identifying such changes in these cancers particularly in the early stages of development, which are not fully known. Identification of these changes is important in developing new treatments as well as markers of behaviour of cancers and also the early or ‘premalignant’ lesions. We used a well-characterised panel of cell lines generated from premalignant lesions as well as cancers, to identify mutations in genes, and an increase or decrease in number of copies of genes. We mapped new and previously identified changes in these cancers to specific stages in the development of these cancers and their spread. We additionally report here for the first time, alterations in CSMD1 gene in early premalignant lesions; we further show that this is likely to result in increased ability of the cells to spread and possibly, multiply faster as well.
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