Absence of keratin 1 restricts the course of infection with lymphocytic choriomeningitis virus strain MX.

A rodent-transmitted enveloped lymphocytic choriomeningitis virus (LCMV) is an RNA virus causing persistent infection. During persistent infection, a unique strain MX of LCMV does not yield infectious virions, therefore it is not able to use a receptor for its dissemination, and spreads by cell-to-cell contacts. Virus can be transported to the neighboring cell by different cellular structures such as tunneling nanotubes or cytonemes. Using q-PCR, immunofluorescence, siRNA and western blot, we show that keratin 1 (K1) is essential for the persistent infection caused by LCMV strain MX, and its absence very effectively slows down the course of infection. In contrast, other LCMV strains, namely Clone 13 and Armstrong, which produce expression of K1, desmosomes in cells expressing K1 (42-MG-BA) but not in cells without K1 expression (NIH/3T3). We conclude that the presence of the virus enhances the K1 expression, while the presence of K1 protein potentiates the viral spread in persistently infected cells. Keywords: lymphocytic choriomeningitis virus; keratin 1; persistent infection; desmosomes; virus transport.

Cell-to-cell transmission of lymphocytic choriomeningitis virus MX strain during persistent infection and its influence on cell migration.

Lymphocytic choriomeningitis virus (LCMV) can establish in its host a persistent infection, without any prominent symptoms. Even during this infection, when the infectious virions are not released, the virus still disseminates effectively. A very effective and fast way of infection of neighboring cells utilized by many viruses is cell-to-cell transmission. Viruses use different ways of cell-to-cell spread through the extracellular space or by intracellular means through different protrusions. We have found that LCMV strain MX may use three different types of cell-to-cell transport. Firstly, similar to vaccinia virus, it can use actin to propel the virus towards the neighboring cell. Secondly, virus can travel through the intracellular space inside the tunneling nanotubes, that connect the cells even at longer distances and thirdly, the virus may travel on the surface of the membrane of different protrusions connecting two cells. We have also proved that the cells infected by MX strain of LCMV migrate faster than the uninfected cells or cells infected with a different LCMV strain. In accordance with faster migration, the infected cells form more lamellipodia with high expression of keratin 1. In this work, we have introduced three types of cell-to-cell transmission utilized by strain MX of LCMV and showed that even if the cells are not in tight connection, the virus forces them to migrate faster to join the nearest cell. As we show in this work, the virus may use more than one strategy to move to another cell, while each strategy can substitute another. These ways of transmission are very fast and effective and may have a serious impact on the host. Moreover, targeting the cell-to-cell spread, by inhibiting for instance GTPase dynamin, could be an effective way of virus elimination. Keywords: lymphocytic choriomeningitis virus; transmission; migration; keratin 1; nucleoprotein.

Lymphocytic choriomeningitis virus: ways to establish and maintain non-cytolytic persistent infection.

Lymphocytic choriomeningitis virus (LCMV) is a prototype virus of the Arenaviridae family that is attracting considerable attention both as an important experimental model system to study acute and persistent viral infections, and as a neglected human pathogen of clinical significance. Notably, LCMV is capable of persisting in an infected host, and escaping the immune system. Here we describe the strategies used by the virus to establish and maintain long-term infection in vitro and/or persistent infection in vivo. We discuss how the viral components (RNA, nucleoprotein, glycoprotein, Z protein) manipulate the host cell machinery to facilitate survival and spread of the virus without disturbing the basal cellular processes. Deep understanding of these strategies is inevitable for the development of approaches towards restricting the virus spread and/or preventing its harmful reactivation. This review summarizes the current status in this area and presents ideas emerging from existing data.

Carbonic anhydrase IX as an anticancer therapy target: preclinical evaluation of internalizing monoclonal antibody directed to catalytic domain.

Carbonic anhydrase IX (CA IX) is a suitable target for various anticancer strategies. It is a cell surface protein that is present in human tumors, but not in the corresponding normal tissues. Expression of CA IX is induced by hypoxia and correlates with cancer prognosis in many tumor types. Moreover, CA IX is functionally implicated in cancer progression as a pro-survival factor protecting cancer cells against hypoxia and acidosis via its capability to regulate pH and cell adhesion. Cancer-related distribution of CA IX allows for targeting cancer cells by antibodies binding to its extracellular domain, whereas functional involvement of CA IX opens the possibility to hit cancer cells by blocking their adaptation to physiologic stresses via inhibition of CA IX enzyme activity. The latter strategy is recently receiving considerable attention and great efforts are made to produce CA IX-selective inhibitor derivatives with anticancer effects. On the other hand, targeting CA IX-expressing cells by immunotherapy has reached clinical trials and is close to application in treatment of renal cell carcinoma patients. Nevertheless, development and characterization of new CA IX-specific antibodies is still ongoing. Here we describe a mouse monoclonal antibody VII/20 directed to catalytic domain of CA IX. We show that upon binding to CA IX, the VII/20 MAb undergoes efficient receptor-mediated internalization, which is a process regulating abundance and signaling of cell surface proteins and has considerable impact on immunotherapy. We evaluated biological properties of the MAb and demonstrated its capacity to elicit anti-cancer effect in mouse xenograft model of colorectal carcinoma. Thus, the VII/20 MAb might serve as a tool for preclinical studies of immunotherapeutic strategies against non-RCC tumors. These have not been explored so far and include broad spectrum of cancer types, treatment of which might benefit from CA IX-mediated targeting.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Cancer-associated carbonic anhydrases and their inhibition.

Cells of the growing tumor tissue are exposed to physiological stresses connected with insufficient delivery of oxygen (hypoxia) and accumulation of acidic products of the glycolytic metabolism (acidosis). Adaptation to these microenvironmental stresses involves remodeling of the cellular expression program mediated by hypoxia-inducible factor (HIF), which activates broad array of genes functionally involved in angiogenesis, anaerobic glycolysis, de-adhesion, invasion etc. This leads to increased aggressiveness of tumors, metastatic spread and poor response to therapy. Genes coding for transmembrane carbonic anhydrase (CA) isoforms IX and XII are induced in response to low oxygen as a part of the hypoxic transcriptome. Moreover, CA IX is a direct target of HIF and serves as a surrogate marker of hypoxia and prognostic indicator. Its expression is strongly linked to different types of tumors with the HIF pathway activated due to genetic defect or physiological hypoxia. CA IX (and possibly also CA XII) is participates in pH regulation, which is important for survival of hypoxic cells. Both enzymes are therefore promising therapeutic molecules targetable by inhibitors of CA activity. Some of these sulfonamide compounds and their derivatives are capable to block CA-mediated pH regulation in hypoxia. This review summarizes research data related to distribution, regulation and functional aspects of CA IX and CA XII, and describes emerging possibilities for clinical exploitation of CA inhibitors as imaging tools and anticancer drugs.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer.

Tumour hypoxia is a microenvironmental factor related to poor response to radiation, chemotherapy, genetic instability, selection for resistance to apoptosis, and increased risk of invasion and metastasis. Hypoxia-regulated carbonic anhydrase IX (CA IX) has been studied in various tumour sites and its expression has been correlated with the clinical outcome. The purpose of this study was to investigate the correlation of CA IX expression with outcome in patients with invasive breast cancer. We conducted a retrospective study examining the effects of carbonic anhydrase IX (CA IX) on survival in patients with breast cancer. To facilitate the screening of multiple tissue blocks from each patient, tissue microarrays were prepared containing between two and five representative samples of tumour per patient. Immunohistochemistry was used to examine expression of CA IX in patients with breast cancer. The study includes a cohort of 144 unselected patients with early invasive breast cancer who underwent surgery, and had CA IX expression and follow-up data available for analysis. At the time of analysis, there were 28 deaths and median follow-up of 48 months with 96% of patients having at least 2 years of follow-up. CA IX was negative for 107 patients (17 deaths) and positive for 37 patients (11 deaths). Kaplan-Meier survival curves show that survival was superior in the CA IX-negative group with a 2-year survival of 97% for negatives and 83% for positives (log-rank test P=0.01). Allowing for potential prognostic variables in a Cox regression analysis, CA IX remained a significant independent predictor of survival (P=0.035). This study showed in both univariate and multivariate analysis that survival is significantly inferior in patients with tumour expressing CA IX. Prospective studies are underway to investigate this correlation in clinical trial setting.

Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours.

AIMS: Carbonic anhydrase (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. The aim of the present study was to investigate CA IX and XII expression in patients with ovarian tumours. METHODS AND RESULTS: A series of ovarian tumours was immunostained for CA IX and XII and the results were correlated with histopathological and clinical parameters. Most cases of borderline mucinous cystadenomas, mucinous cystadenocarcinomas and serous cystadenocarcinomas were moderately or strongly positive for CA IX. In malignant tumours, the staining was most prominent in hypoxic regions. Expression of CA XII was detected in all tumour categories, although the mean staining intensity was weaker than for CA IX in all groups except for clear cell carcinomas. CONCLUSIONS: The wide expression of CA IX and XII in ovarian tumours suggests that these isozymes could represent potential targets in ovarian cancer therapy. The expression pattern of CA IX suggests that it could also serve as a useful histopathological marker protein for hypoxia in malignant ovarian tumours.

Lymphocytic choriomeningitis virus mx strain does not induce the expression of tumor-associated carbonic anhydrase IX in persistently infected HeLa cells.

Summary. - Lymphocytic choriomeningitis virus (LCMV) is an arenavirus that readily causes persistent infections, in which it does not interfere with vital functions of the cells, but can affect expression of "luxurious" genes. MX strain of LCMV (MX LCMV) has been identified as an agent transmissible by cell-to-cell contact in the human carcinoma MaTu cells grown in a mixed culture with HeLa cells. When compared to uninfected HeLa, the MaTu-MX-infected HeLa cells, to which the virus was transmitted via co-cultivation with mitomycin C-treated MaTu cells, showed an elevated expression of a protein called MN, suggesting that MN can be induced by MX LCMV. MN protein was later identified as the carbonic anhydrase isoform IX (CA IX), whose expression has been predominantly associated with hypoxic tumors of poor prognosis. Since the proposal that MX LCMV can induce such a cancer-related protein could substantially change our view on the biology of LCMV-host interaction we undertook its verification. Instead of co-cultivation, we used MaTu cell-free extracts to transmit MX LCMV to HeLa cells. These cells were then grown for more than 30 passages, but the level of MN/CA IX did not increase throughout the whole cultivation period as compared to uninfected HeLa cells. Moreover, a treatment of MaTu-MX-infected HeLa cells with ribavirin eliminated the virus, but did not reduce the MN/CA IX expression. Our results clearly showed that MN/CA IX is independent of MX LCMV and that the virus itself does not influence the MN/CA IX level in HeLa cells.

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

Expression of S100P gene in cervical carcinoma cells is independent of E7 human papillomavirus oncogene.

High-risk human papillomaviruses (HPV) significantly contribute to development of cervical cancer. HPV E7 oncoprotein interferes with the control of cell growth via functional inactivation and/or regulation of multiple molecular targets. Induction of ectopic E7 in breast carcinoma cells has been proposed to decrease transcription of S100P gene, which encodes a calcium-binding protein associated with different types of tumors. We examined a possible relationship between E7 and S100P genes in cervical cell lines. RT-PCR analysis revealed that all HPV-positive cell lines expressed approximately equal levels of E7. Out of them, HeLa, CGL3 and SiHa carcinoma cells as well as HCE16/3 immortalized cells expressed also S100P gene. Inhibition of a DNA methylation by 5-aza-2'-deoxycytidine (5-aza-dC) in S100P-negative cell lines CGL1 and Caski resulted in induced transcription of S100P, but the normal S100P level in SiHa cells was not further increased. Our results suggest that S100P gene expression is independent of E7 in cervical cell lines and that at least in some cases it is subjected to regulation by DNA methylation.

Semi-quantitative RT-PCR assessment of molecular markers in breast large-core needle biopsies.

Large-core needle biopsies are frequently used for the preoperative evaluation of the breast lesions. In addition to initial diagnostic information, they can show the status of molecular markers with predictive and prognostic value and further contribute to an optimal selection of treatment strategy. So far, the potential use of large-core needle biopsies in assessment of marker profile of the breast lesions was studied using the immunostaining approaches. In this work, we sought to determine whether analysis of the large-core needle biopsy by semi-quantitative reverse-transcription polymerase chain reaction reveals molecular data that correspond with the marker status of the subsequently removed tumor. Five molecular markers including ER, PR, c-erbB-2/HER-2/neu, c-erbB-3/HER-3, and CD44 were assessed on mRNA isolated from 23 large-core needle biopsies and corresponding surgical breast cancer specimens using beta2- microglobulin as an internal standard. Significant or highly significant correlation between core biopsies and excised tumors was observed for each marker when the mRNA expression status was scored as positive or negative, with the concordance of the data ranging from 73.9% to 86%. Using the dichotomous scoring, majority of the biopsies (75%) displayed molecular profile that was either identical to the profile of the related tumor specimen, or with the difference in one marker. However, no significant correlation was found when the levels of the markers were expressed as continuous variables, possibly due to intratumoral cell heterogeneity. These results suggest potential usefulness and reliability of semi-quantitative RT PCR in the evaluation of large-core needle biopsies with regard to marker positivity or negativity. On the other hand, the marker-related data expressed as continuous variables cannot be accurately assessed on the large- core needle specimens using this approach, indicating the need for methodological improvements.

Immunotargeting of human cervical carcinoma xenograft expressing CA IX tumor-associated antigen by 125I-labeled M75 monoclonal antibody.

The aim of our present study was to explore a potential use of 125I-labeled murine monoclonal antibody M75 that recognizes carbonic anhydrase IX (CA IX) in the immunotargeting of human cervical carcinoma xenografts in nude mice. CA IX is a hypoxia-inducible antigen, whose expression is significantly associated with carcinomas of the uterine cervix, whereas normal cervical tissue does not express CA IX protein. M75 monoclonal antibody was labeled with 125I and used to quantify hypoxic induction of CA IX expression in vitro in HeLa human cervical carcinoma cells by immunoradiometric assay. HeLa cells showed inducible expression of CA IX in vitro by hypoxia (0.1% O2) and various hypoxia mimicking agents (Co2+, Ni2+, Mn2+, desferrioxamine, o-phenanthroline and Na2S2O4). CA IX expression was also upregulated in the centre of HeLa multicellular clusters (spheroids) corresponding to the conditions of chronic hypoxia. For the immunotargeting study, 125I-M75 was intravenously injected into immunodeficient mice bearing HeLa cervical carcinoma xenografts. Biodistribution profile showed selective and preferential accumulation of 125I-M75 mAb in CA IX expressing HeLa xenografts in comparison with control unreactive 125I-T111 antibody. Specificity was also confirmed by low uptake in CA IX negative C33A xenografts. In addition, CA IX expression in cervical carcinoma xenografts was analyzed by immunohistochemistry with M75. Detailed immunohistochemical analysis of HeLa xenograft sections revealed perinecrotically intensified expression of CA IX. These results indicate that M75 mAb, recognizing CA IX antigen, has targeting properties which could be potentially useful in radioimmunodetection or radioimmunotherapy of human cervical carcinomas and derived metastases.

The hypoxia-inducible genes VEGF and CA9 are differentially regulated in superficial vs invasive bladder cancer.

Regulation by hypoxia may underlie the expression of vascular endothelial growth factor in bladder cancer. We have compared the distribution of vascular endothelial growth factor mRNA with a hypoxia marker, carbonic anhydrase 9 (CA IX). vascular endothelial growth factor mRNA was analysed by in situ hybridisation and CA IX by immunochemistry in 22 cases of bladder cancer. The relationship of microvessels to the distribution of CA IX was determined. In a separate series of 49 superficial tumours, CA IX immunostaining was compared with clinico-pathological outcome. In superficial and invasive disease there was overlap in the expression of vascular endothelial growth factor and CA IX, CA IX being more widespread. Both were expressed predominantly on the luminal surface, and surrounding areas of necrosis (invasive tumours). Expression of both factors was greater in superficial disease. Expression was absent within approximately 80 microm of microvessels. Unlike vascular endothelial growth factor, CA IX did not predict outcome in superficial disease. Differential responses to reoxygenation provide one explanation: vascular endothelial growth factor mRNA declined rapidly, while CA IX expression was sustained for >72 h. Expression of vascular endothelial growth factor mRNA in bladder tumours is consistent with hypoxic regulation and suggests differential regulation in superficial vs invasive disease. The expression of CA IX on the luminal surface justifies investigation of its utility as a therapeutic target/prognostic indicator.

Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours.

BACKGROUND/AIMS: Carbonic anhydrase isoenzyme IX (MN/CA IX) is a transmembrane protein with a suggested function in maintaining the acid-base balance and intercellular communication. Previous studies have demonstrated that MN/CA IX is expressed in the basolateral plasma membrane of normal biliary epithelial cells, but not in hepatocytes. This study was designed to examine the expression of MN/CA IX in hepatobiliary neoplasms and to elucidate its value as a marker for biliary differentiation. METHODS: Fifty-seven hepatobiliary lesions were immunostained for MN/CA IX using biotin-streptavidin complex method. Twenty samples containing normal biliary epithelium and five containing normal liver tissue were used as controls. RESULTS: In the biliary epithelial tumours, immunostaining for MN/CA IX was mainly localized at the basolateral surface of the epithelial cells, like in normal mucosa. All non-invasive dysplastic lesions and 57% of invasive lesions of gall-bladder expressed MN/CA IX. In liver, 78% of cholangiocellular malignant lesions showed a positive reaction for MN/CA IX, whereas only 33% of hepatocellular carcinomas showed a weak immunoreaction. CONCLUSIONS: Our results suggest that abnormal expression of MN/CA IX may be linked to malignant transformation of hepatobiliary cells. In addition, it seems to be a promising marker for biliary differentiation in hepatobiliary neoplasms.

Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.

Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy.

PURPOSE: Carbonic anhydrases are proteins involved in the catalytic hydration of carbon dioxide to carbonic acid. Recent studies show that carbonic anhydrase 9 (CA9) is up-regulated by hypoxia and that its immunohistochemical tissue distribution follows the distribution of the radiosensitizer pimonidazole (C. C. Wykoff et al., Cancer Res. 60: 7075-7083, 2001). Therefore, CA9 expression may show hypoxia levels of clinical importance. EXPERIMENTAL DESIGN: We assessed the expression of CA9 and the microvessel density (MVD; CD31-positive) in 75 locally advanced squamous cell head and neck cancers treated with concurrent chemoradiotherapy with carboplatin. RESULTS: Strong membrane/cytoplasmic CA9 expression, noted in 20/75 (26.6%) tumors, mainly occurred in tumors with very poor vascularization (expression in 63% versus 14%; P < 0.0001), was located around areas of focal necrosis, and was related to poor complete response rate (40% versus 70%; P = 0.02). These observations suggested that CA9 might be a marker of clinically important hypoxia. Combining the CA9 staining and the tumor angiogenicity (MVD), we identified three groups of patients: (a) hypoxic tumors; (b) euoxic highly angiogenic tumors; and (c) euoxic non-highly angiogenic tumors. Groups (a) and (b) had a very poor local relapse-free survival (P < 0.0001). CONCLUSIONS: Stratification of patients undergoing radical radiotherapy using the CA9/MVD model may be useful for the individualization of therapeutic strategies combining antiangiogenesis and hypoxia targeting with radiotherapy.

Differential expression of cytoplasmic carbonic anhydrases, CA I and II, and membrane-associated isozymes, CA IX and XII, in normal mucosa of large intestine and in colorectal tumors.

This study compares the localization of carbonic anhydrase isozymes (CA) I and II and that of IX and XII in normal large intestine and in colorectal tumors. Immunohistochemical studies were performed on 69 colorectal lesions. While the normal mucosa of the large intestine showed high expression for CA I and II, the intensity of the immunostaining for both isozymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pattern of expression observed for these cytoplasmic isozymes and transmembrane CA IX and XII in intestinal tissue specimens supports the suggestion that CA IX and XII may be functionally involved in tumor progression to malignancy and/or in invasion. By contrast, while CA I and II are prominent in normal colorectal mucosa, where they play a role in regulation of pH homeostasis and water and ion transport, loss of expression of these cytoplasmic isozymes consistently accompanies progression to malignant transformation.

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors.

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.