Intracrine vascular endothelial growth factor signaling in survival and chemoresistance of human colorectal cancer cells.

Although the effects of vascular endothelial growth factor (VEGF) on angiogenesis and vascular function are well known, the effects of VEGF on tumor cell function remain to be elucidated. We studied phenotypic changes in human colorectal cancer (CRC) cells with homozygous deletion of VEGF alleles to determine the potential direct role of VEGF on tumor cell function. Loss of VEGF expression led to significantly decreased cell growth and increased spontaneous apoptosis in CRC cells (P<0.01). Loss of VEGF also increased the in vitro sensitivity of cells to the cytotoxic effects of the chemotherapeutic drug 5-fluorouracil, as shown by increased apoptosis (P<0.05). These effects were mediated via upregulation of the proapoptotic mediators caspase-3, cleaved PARP and Bax and downregulation of the pro-survival mediator survivin. Our findings suggest a novel and distinct function of VEGF in mediating autocrine/intracrine CRC cell survival.

CDX2 promotes anchorage-independent growth by transcriptional repression of IGFBP-3.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. We have reported that CDX2 promotes tumorigenicity in a subset of human colorectal cancer cell lines. Here, we present evidence that CDX2 negatively regulates the well-documented growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3). Specifically, CDX2 binds to the IGFBP-3 gene promoter and can repress IGFBP-3 transcription, protein expression and secretion. Furthermore, inhibition of IGFBP-3 partially rescues the decreased anchorage-independent growth phenotype observed in CDX2 knockout cells. These data demonstrate for the first time that (1) CDX2 can function as a transcriptional repressor, and (2) one mechanism by which CDX2 promotes anchorage-independent growth is by transcriptional repression of IGFBP-3.

CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 is a marker of colon cancer, with strong staining in up to 90% of colonic adenocarcinomas. CDX2 heterozygous-null mice develop colonic neoplasms, which have suggested that CDX2 is a tumor suppressor. However, CDX2 has not been reported to affect xenograft growth. Furthermore, CDX2 is rarely mutated in colon cancer, which has led to suggestions that it may play only a minor role as a tumor suppressor in colon cancer. To understand the functional contributions of CDX2 to colon cancer, we disrupted CDX2 in LOVO and SW48 human colon cancer cell lines by targeted homologous recombination. Consistent with the literature, disruption of CDX2 enhanced anchorage-dependent cell proliferation. However, homozygous loss of CDX2 led to significant inhibition of anchorage-independent growth in LOVO cells, and cell lethality in SW48 cells. Further analyses revealed that disruption of CDX2 led to anchorage-independent G1 to S growth arrest and anoikis. In vivo xenograft studies confirmed that disruption of CDX2 inhibited LOVO tumor growth. These data demonstrate that CDX2 mediates anchorage-independent growth and survival. Thus, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.

CDX2 does not suppress tumorigenicity in the human gastric cancer cell line MKN45.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is expressed specifically in the intestine. In mice, ectopic expression of CDX2 in the gastric mucosa gives rise to intestinal metaplasia and in one model, gastric carcinoma. In humans, increased CDX2 expression is associated with gastric intestinal metaplasia and tubular adenocarcinomas. These patterns of expression have shown that CDX2 is important for the initiation of intestinal metaplasia in the gastric mucosa, but the role of CDX2 in established gastric cancer remains unclear. We sought to determine whether CDX2 contributes to tumorigenic potential in established gastric cancer. The CDX2 gene in MKN45 gastric carcinoma cells was disrupted using targeted homologous recombination. The resulting CDX2-/- cells are essentially identical to their parental cells, with the exception of CDX2 ablation. We found no significant differences in the proliferation of CDX2-/- cells compared to CDX2+/+ cells, in vitro or in vivo. Molecular analyses show that loss of CDX2 predominantly altered the expression of genes involved in intestinal glandular differentiation and adhesion. However, there were no microscopic differences in tumor differentiation. We conclude that disruption of CDX2 in MKN45 cells does not significantly affect their tumorigenic potential.

Combination bacteriolytic therapy for the treatment of experimental tumors.

Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors. We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascular compartments of transplanted tumors. Among 26 different strains tested, one (Clostridium novyi) appeared particularly promising. We created a strain of C. novyi devoid of its lethal toxin (C. novyi-NT) and showed that intravenously injected C. novyi-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells. When C. novyi-NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 h, resulting in significant and prolonged antitumor effects. This strategy, called combination bacteriolytic therapy (COBALT), has the potential to add a new dimension to the treatment of cancer.

Expression of the gut-enriched Krüppel-like factor (Krüppel-like factor 4) gene in the human colon cancer cell line RKO is dependent on CDX2.

Gut-enriched Krüppel-like factor (GKLF or KLF4) is a zinc finger-containing, epithelial-specific transcription factor, that functions as a suppressor of cell proliferation. We previously showed that GKLF expression is decreased in intestinal and colonic adenomas, respectively, from multiple intestinal neoplasia (Min) mice and familial adenomatous polyposis (FAP) patients. This study shows that GKLF is induced upon activation of the adenomatous polyposis coli (APC) gene. However, among several human colon cancer cell lines surveyed, expression of GKLF is lowest in RKO, a line with wild-type APC and beta-catenin. RKO contains a mutated allele that encodes the putative tumor suppressor homeodomain protein, CDX2. We show that wild-type CDX2 activates the GKLF promoter and that the mutated CDX2 has a dominant negative effect on wild-type function. Our results may help explain the exceedingly low levels of GKLF expression detected in this cell line, which may in turn contribute to the tumor phenotype.

Decreased expression of the gut-enriched Krüppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients.

Gut-enriched Krüppel-like factor (GKLF) is a zinc finger-containing transcription factor, the expression of which is associated with growth arrest. We compared Gklf expression in intestinal and colonic adenomas to normal mucosa in multiple intestinal neoplasia (Min) mice and familial adenomatous polyposis (FAP) patients, respectively, using semi-quantitative RT-PCR. In Min mice, the level of Gklf transcript is highest in normal-appearing intestinal tissues and decreases as the size of the adenoma increases. In FAP patients, the level of GKLF transcript is lower in adenomas compared to paired normal-appearing mucosa from the same patient or normal colonic mucosa from control individuals without FAP. The possibility of DNA methylation as a cause for the decreased expression of Gklf in adenomas of Min mice was investigated by methylation-specific PCR. Results indicate that the Gklf gene is not methylated in either normal or tumorous tissues. The findings of our study are therefore consistent with the potential role of GKLF as a negative growth regulator of gut epithelial cells.

A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation.

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion.

Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants.

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.

A physical map of the sorghum chloroplast genome.

The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the γbcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.