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

Abstract

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.

Figure 1

Identification and predicted structure of the human splicing variant of CA IX. (A) Schematic illustration of the genomic structure of the human CA9 gene (GenBank no. Z54349). Positions of primers are indicated by arrows. Exons excluded by alternative splicing are in dark grey colour. (B) RT–PCR analysis of CA9 in the human stomach, small intestine, and colon using h1S-h6A primers that do not discriminate between the splicing variants. (C) Amplification of both FL and AS transcripts in the human tissues using h6S-h11A primers. (D) Comparison of amino acid sequences deduced from the human FL and AS CA9 cDNAs. Signal peptide (SP) is written in italic, proteoglycan-like domain (PG) is in bold, carbonic anhydrase domain (CA) is on a grey background, and the transmembrane region (TM) is written by white letters on a black background. Dashed lines represent amino acid residues deleted in AS. Histidines that bind a catalytic zinc and cysteines involved in formation of S-S bonds are placed on a dark background (E) Predicted structure of the human FL and AS CA IX proteins.

Figure 2

Expression of the AS CA IX variant in human tumour cell lines and in human tissues. RT–PCR analysis of human AS CA9 using the primers designed for individual amplification of the splicing variants, namely h7S-h8A for FL and h7S-h10/7A for AS (see Figure 3). β-actin was used as a standard. SmartLadder was included in the right side of the gels. The cDNAs were isolated (A) from the cells exposed to normoxia (N) and hypoxia (H) for 48 h, (B) from the cells incubated at low and high density for 72 h, and (C) from normal and tumour human tissues. The results indicate that the AS expression is steady and does not depend on hypoxia, density, and tumour phenotype.

Figure 3

Localisation of the human AS CA IX. CA IX-negative MDCK cells and HeLa cells with natural hypoxia-induced expression of FL CA IX were permanently transfected with AS CA9 cDNA in pSG5C plasmid. (A) Immunofluorescence analysis of the AS-transfected (AS), FL-transfected (FL), and control cells (mock) was performed using M75 MAb recognising both AS and FL proteins. (B) Immunoblotting analysis of the protein extracts from HeLa-AS and control HeLa cells plated in sparse (S), medium (M), and dense (D) cultures, allowed to adhere for 24 h and then incubated for 24 h in normoxia and hypoxia, respectively. Asterisk on the CA IX blot indicates the position of 50 K Mw marker, α-tubulin was used as a loading control. (C) The AS CA IX variant was also detected with M75 MAb in culture medium of AS-transfected cells.

Figure 4

Effect of overexpressed AS variant on acidification, inhibitor binding, and spheroid formation. (A) The AS-transfected HeLa cells and related mock-transfected controls were incubated for 48 h in normoxia and hypoxia, respectively, and extracellular pH was measured in culture medium immediately at the end of experiment. Data are expressed as differences between the pH values (ΔpH) measured in normoxic vs hypoxic cells and include standard deviations. Results show that expression of AS reduces the acidification mediated by FL CA IX protein under hypoxia. (B) MDCK-CA IX transfected cells that constitutively express human FL CA IX protein were treated for 48 h by a fluorescent CA inhibitor (FITC-CAI) in the absence (control) or in the presence of the secreted AS variant added with the conditioned medium from MDCK-AS transfectants. Conditioned medium was mixed with a fresh cultivation medium. FITC-CAI bound only to hypoxic cells and was considerably reduced in the presence of the AS protein. (C) The same experiment was performed repeatedly with either one-half (1/2 AS) or one-third (1/3 AS) of conditioned medium from MDCK-AS cells. Binding of FITC-CAI and corresponding fluorescence was evaluated from acquired images using Scion Image software. Data were expressed as a percentage of positive control represented by hypoxic MDCK-CA IX cells incubated with FITC-CAI in the absence of AS. The results confirmed that AS reduces the binding of FITC-CAI to CA IX. (D) Microscopic images of spheroids grown from control mock-transfected HeLa cells and from AS-transfected HeLa cells, respectively. Control HeLa cells express hypoxia-induced, functional FL CA IX protein, and produce spheroids that form compact cores. HeLa-AS cells, which contain both hypoxia-induced FL CA IX and constitutively expressed AS, contain loose cores possibly due to AS-compromised function of FL leading to decreased survival of hypoxic core cells.