Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Radioimmunotherapy with a 64Cu-labeled monoclonal antibody: a comparison with 67Cu.

67Cu (t1/2 = 62 h) has demonstrated potential as a radionuclide for radioimmunotherapy, but limited availability severely restricts its widespread use. 64Cu (t1/2 = 12.8 h) has been shown to have comparable effectiveness in vitro and in vivo. The present study was undertaken to examine the therapeutic potential of 64Cu- and 67Cu-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradeca ne-N, N',N",N"'-tetraacetic acid (BAT)-2-iminothiolane (2IT)-1A3 (1A3 is a mouse anti-human colorectal cancer mAb) for treatment of GW39 human colon carcinoma carried in hamster thighs. Hamsters were injected with 64Cu- or 67Cu-BAT-2IT-1A3 or Cu-labeled nonspecific IgG (MOPC) or saline. Hamsters were killed 6-7 months after therapy or when tumors were > or = 10 g. Of the hamsters with small tumors (mean weight 0.43 +/- 0.25 g), 87.5% were disease-free 7 months after treatment with 2 mCi (1 Ci = 37 GBq) of 64Cu-BAT-2IT-1A3 or 0.4 MCi of 67Cu-BAT-2IT-1A3. The mean tumor doses at these activities of 64Cu- and 67Cu-BAT-2IT-1A3 were 586 and 1269 rad (1 rad = 0.01 Gy), respectively. In contrast, 76% of hamsters treated with 2 mCi of 64Cu-BAT-2IT-MOPC or 0.4 mCi of 67Cu-BAT-2IT-MOPC had to be killed before 6 months because of tumor regrowth. When hamsters with larger tumors (mean weight 0.66 +/- 0.11 g) were treated with 64Cu- or 67Cu-BAT-2IT-1A3, survival was extended compared with controls, but only one animal remained tumor-free to 6 months. These results demonstrate that 64Cu- and 67Cu-BAT-2IT-1A3 given in a single administered dose can eradicate small tumors without significant host toxicity, but additional strategies to deliver higher tumor doses will be needed for larger tumors.

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.