Stefan Kaluz

Biodistribution and pharmacokinetics of 125I‐labeled monoclonal antibody M75 specific for carbonic anhydrase IX, an intrinsic marker of hypoxia, in nude mice xenografted with human colorectal carcinoma

Carbonic anhydrase IX (CA IX) is frequently expressed in human carcinomas and absent from the corresponding normal tissues. Strong induction by tumor hypoxia predisposes CA IX to serve as a target for cancer diagnostics and therapy. Here we evaluated targeting properties and pharmacokinetics of CA IX‐specific monoclonal antibody (MAb) M75. Binding parameters of 125I‐labeled M75, including equilibrium dissociation constant, hypoxia‐related binding to various cell lines and internalization, were analyzed in vitro. Biodistribution of 125I‐M75 in nude mice bearing HT‐29 human colorectal carcinoma xenografts with hypoxic pattern of CA IX expression was studied by measurements of radioactivity in dissected tissues and macroautoradiography of tissue sections. Pharmacokinetics of intravenously administered 125I‐M75 was described using a 2‐compartment model. Blood clearance showed a distribution phase t1/2(α) = 3.4 hr and an elimination phase t1/2(β) = 55.3 hr postinjection. Despite predominant CA IX localization in less accessible perinecrotic regions, 125I‐M75 exhibited specific accumulation in xenograft, with a mean uptake of 15.3 ± 3.6% of injected dose per gram of tumor tissue at 48 hr postadministration. Specificity of M75 localization was confirmed by low tumor uptake of control antibody. Altogether, our data demonstrate that M75 MAb is a promising tool for selective immunotargeting of hypoxic human tumors that express CA IX.

Transcriptional Regulation of the MN/CA 9 Gene Coding for the Tumor-associated Carbonic Anhydrase IX IDENTIFICATION AND CHARACTERIZATION OF A PROXIMAL SILENCER ELEMENT

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3.5-kilobase pair MN 5′ upstream region by deletion analysis led to the identification of the −173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa × normal fibroblast hybrid cells.

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.

P53 tumour suppressor modulates transcription of the TATA-less gene coding for the tumour-associated carbonic anhydrase MN/CA IX in MaTu cells

MN/CA IX (MN) exhibits a strong association with tumours. Co-transfection experiments revealed that in MaTu cells the activity of the (-173;+31) MN promoter construct was repressed by the wild type p53 in a dose-responsive manner and stimulated by the (143(Val-->Ala)) mutant. Upregulation of endogenous p53 by mitomycin C treatment in MaTu cells also had a profound effect on MN expression as well as the activity of MN promoter in a reporter construct. p53 can thus modulate MN expression and at least in a subset of tumours the changed p53 status might be responsible for MN positivity. Co-transfections with internally deleted MN promoter constructs demonstrated that the wild type p53 exerts its repression activity on the level of the basal transcriptional machinery and not on a particular cis element within the MN promoter.

Block-replacement mutagenesis for functional dissection of multiple transcription factor complexes

We propose a simple method for investigation of roles of individual transcription factors in complexes of multiple factors bound to the same cis element. By block-replacement mutagenesis, the whole cis element is replaced with a new one containing a binding site for a single factor. From the reporter activity of the mutant promoter construct, the importance of the individual factor in transcriptional activation is deduced. This approach allowed us to functionally identify SP1 as the most important PR1 binding transcription factor in the MN promoter.