Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer.

This corrects the article DOI: 10.1038/onc.2015.296.

CK1δ/GSK3ß/FBXW7α axis promotes degradation of the ZNF322A oncoprotein to suppress lung cancer progression.

Overexpression of Cys2His2 zinc-finger 322A (ZNF322A) oncogenic transcription factor is associated with lung tumorigenesis. However, the mechanism of ZNF322A overexpression remains poorly understood. Here, we discover that protein stability of ZNF322A is regulated by coordinated phosphorylation and ubiquitination through the CK1δ/GSK3ß/FBXW7α axis. CK1δ and GSK3ß kinases sequentially phosphorylate ZNF322A at serine-396 and then serine-391. Moreover, the doubly phosphorylated ZNF322A protein creates a destruction motif for the ubiquitin ligase FBXW7α leading to ZNF322A protein destruction. Overexpression of FBXW7α induces ZNF322A protein degradation, thereby blocks ZNF322A transcription activity and suppresses ZNF322A-induced tumor growth and metastasis in vitro and in vivo. Clinically, overexpression of ZNF322A correlates with low FBXW7α or defective CK1δ/GSK3ß-mediated phosphorylation in lung cancer patients. Multivariate Cox regression analysis indicates that patients with ZNF322A high/FBXW7 low expression profile can be used as an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of ZNF322A oncoprotein destruction regulated by the CK1δ/GSK3ß/FBXW7α axis. Deregulation of this signaling axis results in ZNF322A overexpression and promotes cancer progression.

Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer.

This corrects the article DOI: 10.1038/onc.2015.296.

Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer.

ZNF322A encoding a classical Cys2His2 zinc finger transcription factor was previously revealed as a potential oncogene in lung cancer patients. However, the oncogenic role of ZNF322A and its underlying mechanism in lung tumorigenesis remain elusive. Here we show ZNF322A protein overexpression in 123 Asian and 74 Caucasian lung cancer patients. Multivariate Cox regression analysis indicated that ZNF322A was an independent risk factor for a poor outcome in lung cancer, corroborating the Kaplan-Meier results that patients with ZNF322A protein overexpression had significantly poorer overall survival than other patients. Overexpression of ZNF322A promoted cell proliferation and soft agar growth by prolonging cell cycle in S phase in multiple lung cell lines, including the immortalized lung cell BEAS-2B. In addition, ZNF322A overexpression enhanced cell migration and invasion, whereas knockdown of ZNF322A reduced cell growth, invasion and metastasis abilities in vitro and in vivo. Quantitative proteomic analysis revealed potential ZNF322A-regulated downstream targets, including alpha-adducin (ADD1), cyclin D1 (CCND1), and p53. Using luciferase promoter activity assay combined with site-directed mutagenesis and sequential chromatin immunoprecipitation-PCR assay, we found that ZNF322A could form a complex with c-Jun and cooperatively activate ADD1 and CCND1 but repress p53 gene transcription by recruiting differential chromatin modifiers, such as histone deacetylase 3, in an AP-1 element dependent manner. Reconstitution experiments indicated that CCND1 and p53 were important to ZNF322A-mediated promotion of cell proliferation, whereas ADD1 was necessary for ZNF322A-mediated cell migration and invasion. Our results provide compelling evidence that ZNF322A overexpression transcriptionally dysregulates genes involved in cell growth and motility therefore contributes to lung tumorigenesis and poor prognosis.

[Transcriptome analysis of human breast cancer cell lines MCF-7 and MDA-MB- 435 by RNA-seq].

The transcriptomic profiles of human breast cancer cell lines MCF-7 and MDA-MB-435 were investigated using the next-generation RNA sequencing (RNA-Seq). The DESeq package was used to screen the differentially expressed transcripts. A total of 229 genes with a significantly differential expression in MDA-MB-435 cells as compared with MCF-7 cells were obtained. Annotation of the biological functions of these genes through the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 demonstrated that the 229 differentially expressed genes were mainly implicated in the biological functions related to cell adhesion and motion, antigen processing and presentation (via MHC class II), hormone response, extracellular structure organization, tissue remodeling, and cell proliferation regulation. Analysis of the individual genes demonstrated that MDA-MB-435 cells exhibited a higher tendency to metastasis and antigen processing and presentation, and lower ability to hormone response. Twenty most abundant transcripts in MDA-MB-435 cells, such as VIM, TNC, and CD74, represent its high potential for metastasis. Besides the genes previously reported to be involved in tumor metastasis and development, genes newly identified in this study could provide new clues for the diagnosis and prognosis of aggressive breast cancers.

Endocervical glandular neoplasia associated with lobular endocervical glandular hyperplasia is HPV-independent and correlates with carbonic anhydrase-IX expression: a Gynaecological Oncology Group Study.

BACKGROUND: Lobular endocervical glandular hyperplasia (LEGH) is a rare lesion of the uterine cervix. It has been proposed that LEGH may represent a precursor lesion to a group of mucinous adenocarcinoma with gastric phenotype (GA) that is independent of high-risk human papillomavirus (H-HPV) infection. Carbonic anhydrase-IX (CA-IX) is highly expressed in conventional glandular lesions (CGLs). However, expression of CA-IX in LEGH or GA has not been studied. METHODS: In all, 12 CGLs, 7 LEGHs, 6 LEGHs with coexisting adenocarcinoma in situ (AIS, 3) and GA (3) were identified from Japanese women with a cytological diagnosis of atypical glandular cells of undetermined significance. Immunostaining was used to detect CA-IX and p16(INK)4(a) (hereafter termed p16) protein expression in the tissues and CA-IX protein expression in the Papanicolaou smears (PSs). Polymerase chain reaction was used to detect H-HPV DNA in liquid-based cytology. RESULTS: Out of 12 (83%) CGLs, 10 were positive with H-HPV and high levels of CA-IX expression were seen in all (100%) cases. P16 protein expression was observed in 11 out of 12 (92%) cases. None of the LEGHs, LEGHs with AIS or GA were positive for H-HPV and only 8 out of 13 (62%) showed focal weak (1+) p16 expression. In contrast, all cases (100%) exhibited strong CA-IX protein expression. CONCLUSION: Our study suggests that there are different molecular mechanisms of carcinogenesis resulting in CGLs vs LEGHs associated with AIS or GA. There is also a possible link between LEGHs and GAs. Furthermore, CA-IX expression may serve as a useful biomarker for the detection of GAs in the absence of H-HPV infection.

Changes in the incidence of candidaemia during 2000-2008 in a tertiary medical centre in northern Taiwan.

Candidaemia is associated with high mortality and high healthcare costs. The incidence of candidaemia in Taiwan rose markedly during the period 1980-2000. We conducted this hospital-based surveillance study in order to explore the secular trend in incidence of candidaemia during the period 2000 to 2008. In our study, Candida spp. were the fourth most common cause of bloodstream infections, with a 30-day crude mortality rate of 36.7%. Candida albicans was the most common species identified, although mortality rate did not differ significantly among species. The incidence of candidaemia began to decrease in 2004. Risk factors related to higher mortality included longer hospital stay before onset of candidaemia, liver cirrhosis, malignancy, end-stage renal disease requiring renal dialysis, dependence on mechanical ventilation and urinary catheterisation.

Carbonic anhydrase IX (CA-IX) and high-risk human papillomavirus (H-HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in Japanese women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study.

BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.

Peroxisome proliferator-activated receptor-gamma Pro12Ala polymorphism, Helicobacter pylori infection and non-cardia gastric carcinoma in Chinese.

BACKGROUND: Peroxisome proliferator-activated receptor gamma inhibits the growth and induces apoptosis of gastric cancer cells. A common polymorphism at codon 12 of this gene (Pro12Ala) has been shown to confer protection against diabetes and colorectal cancer. AIM: To study the association between peroxisome proliferator-activated receptor gamma gene polymorphism, Helicobacter pylori infection and gastric cancer in Chinese. METHODS: One hundred and four consecutive patients with non-cardia gastric adenocarcinoma and 104 matched controls were examined. Peroxisome proliferator-activated receptor gamma Pro12Ala polymorphism was analysed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The frequency of peroxisome proliferator-activated receptor gamma G (Ala12) allele was significantly higher among cancer patients (19.2%) than in control (8.7%; OR 2.5, 95% CI 1.1-5.8). While H. pylori infection was more prevalent in gastric cancer patients (OR 3.0; 95% CI 1.6-5.7), the combination of peroxisome proliferator-activated receptor gamma G allele and H. pylori infection further increased the risk of gastric cancer (OR 12.8, 95% CI 3.2-50.5). The presence of the Ala12 did not increase the risk of gastric cancer in H. pylori-negative subjects. CONCLUSION: Our study suggests the potential association between peroxisome proliferator-activated receptor gamma polymorphism and H. pylori infection in the development of non-cardia gastric cancer.

Approximation of flammability region for natural gas-air-diluent mixture.

The growing implementation of exhaust gas recirculation (EGR) in reducing NO(x) emissions of engine is of paramount motivation to perform a fundamental research on the flammability characteristics of fuel-air-diluent mixtures. In this work, the influences of EGR on the flammability region of natural gas-air-diluent flames were experimentally studied in a constant volume bomb. An assumption of critical burning velocity at flammability limit is proposed to approximately determine the flammability region of these mixtures. Based on this assumption, an estimation of the flammability map for natural gas-air-diluent mixtures was obtained by using the empirical formula of burning velocity data. The flammability regions of natural gas-air mixtures with EGR are plotted versus the EGR rate. From the comparison of estimated results and experimental measurements, it is suggested that the accuracy of prediction is largely dependent upon the formula of burning velocity used. Meanwhile, the influence of pressure on the critical burning velocity at flammability limit is also investigated. On the basis of the pressure dependence criterion, the estimation was performed for the circumstance of high temperature and pressure, and the prediction results still agree well with those of experiments.

STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker.

BACKGROUND: STRA13 is a bHLH transcription factor that plays a crucial role in cell differentiation, proliferation, apoptosis, and response to hypoxia. OBJECTIVE: To assess STRA13 involvement in carcinogenesis and evaluate its diagnostic value. METHODS: A comprehensive analysis was undertaken of the endogenous protein expression in 389 normal and corresponding malignant specimens, using newly generated polyclonal antibodies. RESULTS: STRA13 was commonly expressed in epithelial cells of normal and neoplastic tissues where it was confined mostly to the nucleus. Intense cytoplasmic STRA13 immunoreactivity was characteristic of myoepithelial and differentiated squamous epithelial cells of all organ sites and their neoplastic counterparts, suggesting application of STRA13 as a myoepithelial cell marker. A distinctive apical granular cytoplasmic staining pattern observed in the pancreas and large intestine was retained in corresponding metastatic carcinomas, providing for identification of the primary sites of these disseminating tumours. In less differentiated tumours there was a tendency to lose the cytoplasmic staining or to switch to nuclear STRA13 staining. Analysis of STRA13, HIF-1alpha, and CAIX expression patterns in a large set of various tumours substantiated the association of STRA13 with HIF-1alpha expression and hypoxia in vivo. Investigation of the molecular mechanisms of STRA13 nucleo-cytoplasmic shuttling suggested that STRA13 employs nuclear import/export that utilises the NLS/NES motifs situated within the N-terminus and in the middle of the protein. CONCLUSIONS: STRA13 may serve as a marker for myoepithelial cells, for the degree of tumour differentiation, and for identification of the primary site of certain metastatic tumours. In combination with CAIX and CAXII markers, it may lead to a more accurate classification of all renal carcinomas.

Experimental study of flammability limits of natural gas-air mixture.

Flammability limits data are essential for a quantitative risk assessment of explosion hazard associated with the use of combustible gas. The present work is to obtain the fundamental flammability data for prevention of the hazards in the practical applications. Experiments have been conducted in a constant volume combustion bomb, and the fuel considered here is natural gas (NG). The pressure histories in the combustion bomb are recorded and a criterion of 7% pressure rise has been used to judge a flammable mixture. The effects of ethane on NG-air flammability limits have been investigated. By adding diluent (carbon dioxide, nitrogen or their mixture) into NG-air mixture, the dilution effects on the flammability limits have been explored as well, and the results are plotted as functions of diluent ratio.

Intratumoral oxygenation of invasive squamous cell carcimoma of the vulva is not correlated with regional lymph node metastasis.

INTRODUCTION: Tumour hypoxia has been found to be associated with tumour aggressiveness. Our primary aim was to explore the relationship between pretreatment tumour oxygenation in primary vulvar carcinoma and nodal status. Our secondary objective was to assess if there was a relationship between the clinical and biological variables. METHODS: 20 women with ISCC of the vulva were assessed with pretreatment primary tumour oxygenation with an Eppendorf pO2 probe. Patients underwent standard surgical management. Pathological assessment of the primary and nodal tissues was then performed. Primary tumour specimens were also stained for microvessel density and carbonic anhydrase IX. The relationship between smoking, preoperative Hgb, tumour CAIX expression, MVD, and Eppendorf pO2 measurements vs nodal metastasis and between these clinical and biological variables was assessed. RESULTS: Seven patients had positive lymph nodes, 13 had negative nodes. While neither current smoking status, tumour size, tumour oxygen measurements, MVD and CAIX expression correlated with metastatic nodal disease, a low preoperative Hgb correlated with pathological nodal status (p < 0.027). CONCLUSIONS: Although this analysis failed to demonstrate a strong correlation between various measures of tumour oxygenation with nodal metastasis, it may be due to the small number of patients. Only preoperative anaemia is correlated with nodal metastasis in early ISCC of the vulva.

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma.

PURPOSE: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma. METHODS: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors. RESULTS: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots. CONCLUSIONS: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer.

The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.

Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer.

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Breast cancer, passive and active cigarette smoking and N-acetyltransferase 2 genotype.

The relationship of breast cancer to cigarette smoking is inconsistent in the literature, possibly due in part to heterogeneity in carcinogen metabolism. N-acetyltransferase 2 (NAT2) enzyme activity is believed to play a role in the activation of tobacco smoke carcinogens. We examined the effect of NAT2 genetic polymorphisms on risk of breast cancer from active and passive smoking. Women were recruited from those who had suspicious breast masses detected clinically and/or mammographically. Questionnaire data were collected prior to biopsy diagnosis to blind subjects and interviewers. Histopathology showed 113 cases with mammary carcinoma (30 carcinoma in situ) and 278 controls with benign breast disease. NAT2 genotype was determined using allele-specific polymerase chain reaction amplification to detect slow acetylator mutations. Effects of passive and active tobacco smoke and of NAT2 genotype on breast cancer risk were examined with logistic regression controlling for known risk factors. Models first included all controls, and subsequently 107 with no or low risk (normal breast or no hyperplasia), and finally 148 with high risk (hyperplasia, atypical hyperplasia, complex fibroadenomas). Referents had no active or passive smoke exposure. We found no association between breast cancer risk and NAT2, smoking status (never, former, current), smoking duration, or cigarettes per day. There were no effects of passive exposure among never-smokers. Models were unchanged across control groups. There were no statistical interactions between tobacco smoke exposure and NAT2. The results were similar when restricting the analysis to invasive cancers. These findings do not support the hypothesis that NAT2 is a risk factor for breast cancer or that it alters susceptibility to tobacco smoke.

Breast cancer, heterocyclic aromatic amines from meat and N-acetyltransferase 2 genotype.

Breast cancer risk has been hypothesized to increase with exposure to heterocyclic aromatic amines (HAAs) formed from cooking meat at high temperature. HAAs require enzymatic activation to bind to DNA and initiate carcinogenesis. N-acetyltransferase 2 (NAT2) enzyme activity may play a role, its rate determined by a polymorphic gene. We examined the effect of NAT2 genetic polymorphisms on breast cancer risk from exposure to meat by cooking method, doneness and estimated HAA [2-amino-1-methyl-6-phenylimidazole[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)] intake. Women were recruited with suspicious breast masses and questionnaire data were collected prior to biopsy to blind subjects and interviewers to diagnoses. For 114 cases with breast cancer and 280 controls with benign breast disease, NAT2 genotype was determined using allele-specific PCR amplification to detect slow acetylator mutations. HAAs were estimated from interview data on meat type, cooking method and doneness, combined with a quantitative HAA database. Logistic regression models controlled for known risk factors, first including all controls, then 108 with no or low risk (normal breast or no hyperplasia) and finally 149 with high risk (hyperplasia, atypical hyperplasia, complex fibroadenomas). Meat effects were examined within NAT2 strata to assess interactions. We found no association between NAT2 and breast cancer. These Californian women ate more white than red meat (control median 46 versus 8 g/day). There were no significant associations of breast cancer with red meat for any doneness. White meat was significantly protective (>67 versus <26 g/day, OR 0.46, 95% CI 0.23-0.94, P for trend = 0.02), as was chicken, including well done, pan fried and barbecued chicken. MeIQx and DiMeIQx were not associated with breast cancer. A protective effect of PhIP was confounded after controlling for well done chicken. Results were unchanged using low or high risk controls or dropping 30 in situ cases. There was no interaction between NAT2 and HAAs. These findings do not support a role for HAAs from meat or NAT2 in the etiology of breast cancer. Further research is needed to explain the white meat association.

Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine-human xenograft model.

OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.

Expression of MN/CA9 protein in Papanicolaou smears containing atypical glandular cells of undetermined significance is a diagnostic biomarker of cervical dysplasia and neoplasia.

BACKGROUND: Despite the enormous impact that Papanicolaou (Pap) smear screening has had on the incidence of cervical carcinoma in developed countries, there is still an unacceptably high frequency of occurrence of this cancer. In part, this is due to human error associated with cytologic diagnoses of Pap smears. Also, the use of new sampling devices, such as the cytobrush, has increased the complexity of diagnosing benign and neoplastic cervical cytology. This is particularly apparent in the diagnosis of atypical glandular cells of undetermined significance (AGUS). Approximately 40% of AGUS diagnoses have a corresponding significant lesion at biopsy follow-up, and 60% do not. There is clearly a need for an adjunct to cytologic diagnosis that can readily identify AGUS smears that are diagnostic of significant lesions. The authors have identified the MN/CA9 antigen as a strong candidate for an adjunct biomarker. METHODS: A total of 245 Pap smears of all AGUS diagnostic categories with histologic confirmation were studied. The median age of the patients was 39 years. The Bethesda system classification (AGUS-favor reactive, AGUS-not otherwise specified, and AGUS-favor neoplastic) was used. All of the Pap smears were decolorized and immunostained with monoclonal antibody to MN/CA9 antigen by the immunoperoxidase technique. The results of MN/CA9 immunoreactivity were correlated with the histologic data in a semiblinded fashion. RESULTS: The follow-up biopsies showed that a high percentage (70%) of patients had low and high grade cervical intraepithelial neoplasia lesions, respectively (CIN I and CIN II or III). Clinically significant lesions-adenocarcinoma in situ/carcinoma (AIS/CA) and CIN II or III-were found in 50% of the cases. Among these, 11% were AIS/CA. In the three subcategories of AGUS diagnosis, the AGUS-not otherwise specified showed the broadest range of lesions in the follow-up biopsies. Three patterns of MN/CA9 immunoreactivity were observed in the Pap smears: 1) atypical cells, 2) normal endocervical cells only, and 3) all cells negative. All Pap smears that were MN/CA9 positive were histologically confirmed to be clinically significant lesions or CIN I; in addition, there were a very small number (n = 12) of cases of atypia. None of the benign lesions showed MN/CA9 expression in the corresponding Pap smears. Furthermore, the pattern of atypical cell immunostaining identified all cases with significant lesions (AIS/CA and CIN II or III) in the cervices. Conversely, the majority of CIN I cases (82%) and all cases of atypia showed positive immunostaining restricted to normal endocervical cells only. CONCLUSIONS: There is a clear association between MN/CA9 immunostaining of atypical cells and the presence of significant lesions in the cervix. Similarly, there is a clear association between lack of expression of MN/CA9 and the absence of cervical lesions. However, the screen does not allow discrimination between CIN I and atypia. The authors also found that, based on the combined patterns of morphology and immunostaining, they are able to discriminate between AIS and CIN II or III in AGUS Pap smear diagnoses. Thus, expression of the MN/CA9 antigen is indeed a discriminator of significant lesions in AGUS Pap smear diagnoses.