The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21^Cip1 and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21^Cip1 and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21^Cip1 expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21^Cip1 expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21^Cip1 core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21^Cip1 promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21^Cip1 expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21^Cip1 expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21^Cip1 gene expression.     

The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.     

The human papillomavirus type 16 E5 protein alters vacuolar H(+)-ATPase function and stability in Saccharomyces cerevisiae

The human papillomavirus 16 (HPV-16) E5 oncoprotein is a small integral membrane protein that binds to the 16-kDa subunit of the vacuolar H(+)-ATPase (v-ATPase). Conservation within the family of v-ATPases prompted us to look to Saccharomyces cerevisiae as a potential model organism for E5 study. The E5 open reading frame, driven by a galactose-inducible promoter, was integrated into the yeast genome, and the resulting strain demonstrated a nearly complete growth arrest at neutral pH, consistent with defects associated with yeast v-ATPase mutants. Furthermore, this strain demonstrated a severe reduction in pH-dependent and v-ATPase-dependent vacuolar localization of fluorescent markers. Overexpression of the yeast 16-kDa subunit homolog partially suppressed E5-associated growth defects. E5 expression was correlated with a disassociation of the integral (V(o)) and peripheral (V(i)) v-ATPase sub-complexes, as well as a dramatic reduction of the steady-state levels of one mature V(o) subunit and the concomitant accumulation of its major proteolytic fragment, with unchanged levels of two V(i) subunits. Similar analyses of selected E5 mutants in yeast demonstrated a correlation between E5 biology and v-ATPase disruption. Our observations suggest that wild-type HPV-16 E5 acts during the assembly of the v-ATPase to inhibit, either directly or indirectly, V(o) stability and complex formation.     

Expression of Pirh2, a p27(Kip1) ubiquitin ligase, in hepatocellular carcinoma: correlation with p27(Kip1) and cell proliferation

p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.     

Inverse expression of Jun activation domain binding protein 1 and cell cycle inhibitor p27Kip1: influence on proliferation in hepatocellular carcinoma

Recently, the functional role of Jun activation domain binding protein 1 (Jab1) as a putative novel oncogene in hepatocellular carcinoma (HCC) has been postulated. We show that expression of p27(Kip1), a negative cell cycle regulator, correlates inversely with Jab1 expression in HCC (P = .014). We observed nuclear Jab1 expression in 57% (55/97) and p27(Kip1) expression in 32% (31/97) of HCCs. Neither Jab1 nor p27(Kip1) nor inverse Jab1 and p27(Kip1) expression correlated with clinicopathological parameters. However, HCCs lacking p27(Kip1) with increased proliferative activity were frequently found to express Jab1 (P = .048). Normal liver tissue, cirrhosis, and tumor-like lesions (focal nodular hyperplasia, dysplastic nodules in cirrhotic liver) showed no significant Jab1 expression. In transfection studies in the hepatoma cell line Huh 7, Jab1 overexpression resulted in reduced p27(Kip1) protein levels. We conclude that Jab1 expression may lead to down-regulation of the negative cell cycle regulator p27(Kip1), pointing to a possible mechanism that promotes hepatocarcinogenesis.     

HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1

Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.     

Human papillomavirus type 45 E7 is a transforming protein inducing retinoblastoma protein degradation and anchorage-independent cell cycle progression

High-risk human papillomaviruses (HPV) cause cervical cancer. The biological properties of HPV-45, the third most prevalent high-risk HPV-genotype, are unknown. We demonstrate here that the HPV-45 E7 protein transforms immortalized NIH3T3 fibroblasts, while mutations in either the conserved LXCXE sequence (C28G) or the carboxyl-terminus (Δ87LQQLF91) significantly abolish this activity. To address the mechanisms underlying cell transformation by HPV-45 E7, we investigated its impact on the cell cycle. We show that HPV-45 E7 associates with the hypophosphorylated form of the retinoblastoma protein (pRb) and induces a significant reduction in the pRb half-life which can be blocked by epoxomicin. Moreover, HPV-45 E7 induces anchorage-independent cell cycle progression of NIH3T3 cells and extends the lifespan of primary human keratinocytes. HPV-45 E7C28G did not bind pRb and could neither induce pRb-proteolysis nor promote cell cycle progression. HPV-45 E7Δ87LQQLF91 had intermediate pRb-binding affinity and retained a residual activity to induce the degradation of pRb but lost the capability to promote cell cycle progression in suspension. Another carboxyl-terminal mutant, HPV-45 E7Δ81AEDL84, showed a trend to reduced transforming activity, had reduced pRb-binding activity and lost the capability to induce pRb-degradation; however, this mutant could induce anchorage-independent cell cycle progression with the same efficiency as HPV-45 E7 wild type. In summary, these data suggest that HPV-45 E7 is a transforming protein and that abrogation of cell cycle control contributes to its oncogenic potential.     

p27Kip1反义寡核苷酸对B淋巴瘤细胞WEHI-231细胞周期的影响

目的　探讨p2 7Kip1在抗原受体介导的B淋巴瘤细胞WEHI- 2 31细胞周期停止信号中的作用。方法　用抗IgM抗体诱导WEHI- 2 31细胞细胞周期停止。用合成的p2 7Kip1反义寡核苷酸抑制p2 7Kip1基因的表达。采用流式细胞仪 ,分析细胞核的DNA含量和细胞周期的变化。用体外激酶实验检测Cdk2的活性 ,Westernblot检测Rb蛋白的磷酸化水平。结果　合成的p2 7Kip1反义寡核苷酸能阻断抗IgM抗体诱导的p2 7Kip1基因表达的上调。用p2 7Kip1反义寡核苷酸处理WEHI-2 31细胞 ,可恢复抗原受体交联引起的周期素依赖性激酶Cdk2活性的降低 ,以及Rb蛋白磷酸化水平的下降 ,并使细胞周期恢复运转。结论　p2 7Kip1可能在抗原受体信号介导的WEHI-2 31细胞的细胞周期停止中发挥主要作用     

Differential modulation of cyclin-dependent kinase inhibitor p27Kip1 by negative signaling via the antigen receptor of B cells and positive signaling via CD40

The cross-linking of surface immunoglobulins (sIg) of B cells can transmit a negative signal, resulting in cell cycle arrest, apoptosis or both. Signaling via the B cell antigen CD40 reverses the sIg-mediated negative signaling and induces activation and proliferation of B cells. We investigated the molecular mechanisms for cell cycle regulation by negative and positive signaling via sIg and CD40, respectively, by using the B cell line WEHI-231. Cross-linking of sIg almost completely reduced the activity of cyclin-dependent kinase (Cdk) 2, essential for cell cycle progression in the late G1 phase, although the level of Cdk2 was not reduced. Among the factors that regulate Cdk2 activation, the activity of the Cdk-activating kinase (CAK) appeared intact and cyclin E was reduced only partially in sIg-cross-linked WEHI-231. In contrast, sIg cross-linking induced a significant Cdk inhibitor (CKI) activity. Since a 27-kDa protein was co-precipitated with Cdk2 in anti-Ig-treated, but not untreated WEHI-231, and the CKI activity in anti-Ig-treated WEHI-231 was neutralized by anti-p27^Kip1, antibodies, it is most likely that p27^Kip1 is responsible for the CKI activity induced by sIg cross-linking. p27^Kip1 may thus play a role in growth inhibition of B cells by negative signaling via sIg. In contrast, CD40 signaling enhanced Cdk2 activity and reduced the p27^Kip1 level in anti-Ig-treated WEHI-231, suggesting that the reduction of p27^Kip1 plays an important role in the abrogation of sIg-mediated growth arrest by CD40 signaling. Taken together, p27^Kip1 is likely to be a crucial target molecule of the negative signaling via sIg and the positive signaling via CD40 essential for T cell-dependent immune responses.     

The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway

E7, the major transforming protein of high-risk human papillomavirus (HPV), type 16, binds and inactivates the retinoblastoma protein (pRb), and the Rb-related proteins p107 and p130. HPV16 E7 is a nuclear protein lacking a classical basic nuclear localization signal. In this study we investigated the nuclear import of HPV16 E7 oncoprotein in digitonin-permeabilized HeLa cells. HPV16 E7 nuclear import was independent of pRb, as an E7(DeltaDLYC) variant defective in pRb binding was imported into the nuclei of digitonin-permeabilized cells as efficiently as wild-type E7 in the presence of exogenous cytosol. Interestingly, we discovered that HPV16 E7 is imported into the nuclei via a novel pathway different from those mediated by Kap alpha2beta1 heterodimers, Kap beta1, or Kap beta2. Nuclear accumulation of E7 required Ran and was not inhibited by the RanG19V-GTP variant, an inhibitor of Kap beta mediated import pathways. Together the data suggest that HPV16 E7 translocates through the nuclear pores via a nonclassical Ran-dependent pathway, independent of the main cytosolic Kap beta import receptors.     

人乳头瘤病毒16型E5转化基因的克隆及原核表达

目的建立人乳头瘤病毒16型E5基因的无性繁殖系,为进一步研究其致癌机理作准备.方法用PCR技术从HPV16质粒DNA中扩增出E5基因序列,连接到pGEM-T载体,将阳性的重组pGEM-T用BamH Ⅰ/EcoRⅠ双酶切并回收目的片段连接到原核表达载体pGEX-2T,转化DH5α菌株.取工程菌,IPTG诱导表达,SDS-PAGE电泳鉴定.结果①经PCR、测序和双酶切鉴定,认为HPV16E5正确插入上述表达载体,建立了该转化基因的无性繁殖系.②经IPTG诱导的pGEX-2T-E5质粒表达出约42KD的融合蛋白,分析含有约16KD HPV16 E5蛋白,与预期含HPV16 E5的融合蛋白分子量相符.结论①采用PCR克隆技术,扩增HPV16 E5转化基因目的片段,将其克隆到pGEM-T载体在国内首次建立了HPV16 E5转化基因的无性繁殖子.②成功构建HPV16 E5基因的原核表达质粒,并表达出GST-E5融合表达蛋白.     

Expression of the human papillomavirus type 16 E7 oncoprotein induces an autophagy-related process and sensitizes normal human keratinocytes to cell death in response to growth factor deprivation

Expression of oncogenes, such as the human papillomavirus type 16 (HPV16) E7 oncoprotein, promotes aberrant cell proliferation. In the absence of concurrent mitogenic stimuli, this triggers a cell-intrinsic defense mechanism, the “trophic sentinel response”, which eliminates such aberrant cells. The molecular pathways that elicit this response, however, remain obscure. We set up an experimental system to investigate the trophic sentinel pathway triggered by HPV16 E7 expression in normal human keratinocytes, the natural host cells of HPVs. Keratinocytes expressing HPV16 E7 cultured in E-medium undergo cell death and show increased sub-G1 DNA content when grown to confluence or under conditions of serum deprivation. Moreover, HPV16 E7 expressing human keratinocytes express higher levels of the autophagy marker, LC3-II, which can be abrogated by 3-methyladenine, an autophagy inhibitor. These findings indicate that even under normal culture conditions, HPV16 E7 expression triggers metabolic stress that may result in autophagy, a pathway implicated in carcinogenesis.     

Suppressor activity of anergic T cells induced by IL-10-treated human dendritic cells: association with IL-2- and CTLA-4-dependent G1 arrest of the cell cycle regulated by p27Kip1

We have previously shown that human IL-10-treated dendritic cells (DC) induce an antigen-specific anergy in CD4+ T lymphocytes. These anergic T cells are characterized by an inhibited proliferation, a reduced production of IL-2, and additionally display antigen-specific suppressor activity. In this study we investigated the mechanisms underlying the anergic state and regulatory function of these T cells. We did not observe enhanced rates of programmed cell death of anergic CD4+ suppressor T cells compared to T cells stimulated with mature DC. Cell cycle analysis by DNA staining and Western blot experiments revealed an arrest of anergic CD4+ T suppressor cells in the G1 phase. High levels of the IL-2-dependent cyclin-dependent kinase (cdk) inhibitor p27Kip1 were found in anergic CD4+ suppressor T cells resulting in an inhibited activation of retinoblastoma protein and an arrest of cell cycle progression in the G1 phase. Addition of IL-2, but not blocking of the CTLA-4 pathway restored the proliferation of the suppressor T cells. In contrast, both treatments induced a down-regulation of p27Kip1 and acomplete inhibition of the antigen-specific regulatory function as demonstrated by high proliferation and enhanced IFN-gamma production of co-cultured T cells. Further experiments demonstrated that p27Kip-expressing regulatory CD4+CD25+ T cells did not contribute to induction of T cell anergy in this model. Our data show that regulatory function of anergic CD4+ suppressor T cells is associated with an arrest in the G1 phase of the cell cycle mediated by increased levels of the IL-2- and CTLA-4-dependent cdk inhibitor p27Kip1.     

Rho GTPases and cell cycle control

Members of the Rho family of small GTPases are crucial regulators of biological responses in eukaryotic cells, including cytoskeletal dynamics, cell motility and cell cycle progression. In the present review, we summarize our current understanding of the role of Rho proteins in cell cycle control, highlighting the contribution of specific members of the Rho family and their downstream targets to the regulation of key elements from the core cell cycle machinery, mostly involved in the G1/S transition.     

P16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix

Carcinogenesis of cervical cancer has been investigated, and p16(INK4a) overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16(INK4a) overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16(INK4a) is overexpressed in small cell carcinoma, and if p16(INK4a) is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16(INK4a) overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16(INK4a) was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16(INK4a) was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma.     

Subcellular localization of p27 and prostate cancer recurrence: automated digital microscopy analysis of tissue microarrays

Previous investigations have linked decreased nuclear expression of the cell cycle inhibitor p27 with poor outcome in prostate cancer. However, these reports are inconsistent regarding the magnitude of that association and its independence from other predictors. Moreover, cytoplasmic translocation of p27 has been proposed as a negative prognostic sign. Given the cost and accuracy limitations of manual scoring, particularly of tissue microarrays, we determined if laser-based fluorescence microscopy could provide automated analysis of p27 in both nuclear and cytoplasmic locations and, thus, clarify its significance as a prognostic biomarker. We constructed tissue microarrays covering 202 recurrent cases (rising prostate-specific antigen) and 202 matched controls without recurrence. Quadruplicate tumor samples encompassed 5 slides and 1616 cancer histospots. Cases and controls matched on age, Gleason grade, stage, and hospital. We immunolabeled epithelial cytoplasm with Alexafluor 647, p27 with Alexafluor 488, and nuclei with 4c6-diamidino-2-phenylindole·2HCl. Slides were scanned on an iCys laser scanning cytometer (CompuCyte Corp, Cambridge, MA). Nuclear crowding required a stereological approach--random arrays of circles (phantoms) were layered on images and the content of each phantom was analyzed in scatter plots. Both nuclear and cytoplasmic p27 were significantly lower in cases versus controls (P = .014 and P = .004, respectively). Regression models controlling for matching variables plus prostate-specific antigen showed strong linear trends for increased risk of recurrence with lower p27 in both nucleus and cytoplasm (highest versus lowest quartile; odds ratio, 0.35; P = .006). Manual scoring identified an inverse association between p27 expression and tumor grade but no independent association with recurrence. In conclusion, we developed an automated method for subcellular scoring of p27 without the need to segment individual cells. Our method identified a strong relationship, independent of tumor grade, stage, and prostate-specific antigen, between p27 expression--regardless of subcellular location--and prostate cancer recurrence. This relationship was not observed with manual scoring.     

Prognostic implications of cyclin B1, p34cdc2, p27(Kip1) and p53 expression in gastric cancer

PURPOSE: Cell cycle progression is regulated by interactions of specific cyclins and cyclin dependent kinases (CDKs) at the G1-S and G2-M checkpoints and cell cycle deregulation plays a major role in carcinogenesis of human cancers. PATIENTS AND METHODS: To investigate the role of cell cycle regulators in the pathogenesis and progression of human gastric cancers, 23 cases of gastric carcinomas were examined for the expression of cyclin B1, p34cdc2, p27(Kip1) and p53 by immunohistochemical methods, and gene expression was correlated with various clinicopathologic findings. RESULTS: Out of 23 cases studied, cyclin B1 was diffusely expressed in 20 cases (87.0%), p34cdc2 in 14 cases (60.9%) and p53 in 12 cases (52.2%), whereas in normal gastric tissues, cyclin B1 and p34cdc2 were weakly expressed and p53 was not expressed. In contrast, p27(Kip1) was expressed in only 8.7% of gastric carcinomas compared with 78.3% of normal gastric tissues. There was correlation between the expression of cyclin B1 and expression of p34cdc2 (p=0.002), between the expression of cyclin B1 and loss of p27(Kip1) (p=0.025), and between the expression of p34cdc2 and loss of p27(Kip1) (p=0.065). In addition, expression of cyclin B1 was correlated with regional lymph node metastasis (p=0.032). CONCLUSION: Our results indicate that cyclin B1 and p34cdc2 are involved in the genesis or progression of gastric cancers. Furthermore, overexpression of cyclin B1 may play an important role in lymph node metastatic potential of gastric cancer. Thus, abnormal expression of cyclin B1 and CDKs, overexpression of p53 and loss of p27(Kip1) expression may play important roles in human gastric carcinogenesis.     

The role of the human papillomavirus type 18 E7 oncoprotein during the complete viral life cycle

The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the viral life cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the viral life cycle. In this study, we investigated the role of HPV18 E7 in the viral life cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the viral life cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the viral life cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the viral life cycle.     

Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ

E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.