HPV16 E6/E7基因及蛋白表达与宫颈癌的相关性研究

目的研究人乳头状瘤病毒16型（HPV16）E6／E7基因及其蛋白表达在宫颈疾病及其癌变中的作用。方法运用PCR技术检测51例宫颈癌（癌症组）、20例富颈上皮瘤变（CIN）Ⅱ～Ⅲ级（CIN组）、20例宫颈炎（炎症组）患者病变组织中HPV16 E6／E7基因,并运用免疫组化SP法检测癌症组癌组织中HPV16E6、E7的表达情况。结果癌症组、CIN组、炎症组HPV16 E6检出率分别为5％、35％、45％．后两者明显高于前者（P〈0．05）,HPV16 E7检出率分别为65％、75％、68．6％,P均〉0．05;癌症组45例HPV16 E6和42例E7蛋白阳性表达（88．2％、82．3％）。HPV16 E6蛋白表达与临床分期、肿瘤分化程度和淋巴结有无转移均无相关性（P〉0．05）,HPV16 E7蛋白表达与临床分期和淋巴结有无转移相关（P〈0．05）,与肿瘤分化程度无相关性（P〉0．05）。结论HPV16 E6／E7基因与宫颈疾病及其癌变的关系密切。     

ADP-ribosylation factor is functionally and physically associated with the Golgi complex.

ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.     

FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.     

Molecular chaperones and the process of cellular immortalization in vitro

It is suggested that the chaperone-activity of the SV40 T protein is responsible for the first step in the process of cellular immortalization in vitro: the extension of life span of most transfected cells. Further that an additional up-regulation of selected molecular chaperones is causal in the second step of the process: the rare acquisition of an unlimited cell division potential. Possibly the molecular chaperones are evolution facilitators, enabling the immortalization of primary cells in vitro, as well as the evolution of longevity in species. This revised version was published online in July 2006 with corrections to the Cover Date.     

E6/E7 Functional Differences among Two Natural Human Papillomavirus 18 Variants in Human Keratinocytes

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants’ functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.     

A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding

VP40, the putative matrix protein of both Ebola and Marburg viruses, possesses a conserved proline-rich motif (PY motif) at its N terminus. We demonstrate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) function. In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins that have type I WW-domains, including the mammalian ubiquitin ligase, Nedd4. Single point mutations that disrupted the PY motif of VP40 abolished the PY/WW-domain interactions. Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4. The VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay. These data demonstrate that the VP40 protein of Ebola virus possesses a PY motif that is functionally similar to those described previously for Gag and M proteins of specific retroviruses and rhabdoviruses, respectively. Last, these studies imply that VP40 likely plays an important role in filovirus budding, and that budding of retroviruses, rhabdoviruses, and filoviruses may proceed via analogous mechanisms.     

HPV E6/E7蛋白在肺癌中的研究进展

肺癌是一种多因素参与的恶性肿瘤性疾病.目前在肺癌中检测到HPV E6/E7蛋白存在的报道不断出现,其在肺癌的作用也不断引起研究者的关注.本文就HPV E6/E7蛋白的生物特性与发病机制,以及在肺癌中所起的作用作一综述.     

The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na(+) and K(+) homeostasis and plant tolerance to high Na(+) and low K(+) environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis indicates that SOS2 and SOS3 function in the same pathway. Biochemically, SOS2 interacts with SOS3 in the yeast two-hybrid system and in vitro binding assays. The interaction is mediated by the C-terminal regulatory domain of SOS2. SOS3 activates SOS2 protein kinase activity in a Ca(2+)-dependent manner. Therefore, SOS3 and SOS2 define a novel regulatory pathway important for the control of intracellular ion homeostasis and salt tolerance in plants.     

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.     

HPV16/18E6和P53基因蛋白在喉鳞癌中的表达及其相关性研究

目的探讨人乳头瘤病毒16/18型早期蛋白E6(HPV16/18 E6)、P^53基因蛋白在喉鳞癌(LSCC)中的表达及其相关性.方法免疫组化SABC法检测HPV16/18E6和P^53基因蛋白在LSCC及声带息肉中的表达.结果 HPV16/18E6在声带息肉中未见表达,在LSCC中的表达率为40.0%,差异有显著性(P＜0.05). P^53基因蛋白在声带息肉与LSCC中的表达率分别为6.66%和66.2%,差异有显著性(P＜0.05).HPV16/18E6及 P^53基因蛋白的表达与LSCC临床分期、淋巴结转移有关.二者的表达无明显相关性.结论 HPV16/18型感染和P^53基因异常与LSCC发生、发展有关.HPV16/18E6与P^53基因功能异常无明显相关性.     

Identification of High-Risk Human Papillomavirus DNA,p16, and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.