DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2⋅hMSH6) and hMutSβ (hMSH2⋅hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base–base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSα restores base–base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

Guanylyl cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues

Guanylyl cyclase C (GCC) has been detected only in intestinal mucosa and colon carcinoma cells of placental mammals. However, this receptor has been identified in several tissues in marsupials, and its expression has been suggested in tissues other than intestine in placental mammals. Selective expression of GCC by colorectal tumor cells in extraintestinal tissues would permit this receptor to be employed as a selective marker for metastatic disease. Thus, expression of GCC was examined in human tissues and tumors, correlating receptor function with detection by PCR. GCC was detected by ligand binding and catalytic activation in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues or tumors. Similarly, PCR yielded GCC-specific amplification products with specimens from normal intestine and primary and metastatic colorectal tumors, but not from extraintestinal tissues or tumors. Northern blot analysis employing GCC-specific probes revealed an ≈4-kb transcript, corresponding to recombinant GCC, in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues. Thus, GCC is selectively expressed in intestine and colorectal tumors in humans and appears to be a relatively specific marker for metastatic cancer cells in normal tissues. Indeed, PCR of GCC detected tumor cells in blood from some patients with Dukes B colorectal cancer and all patients examined with Dukes C and D colorectal cancer, but not in that from normal subjects or patients with Dukes A colon carcinoma or other nonmalignant intestinal pathologies.

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.

Prostacyclin-mediated activation of peroxisome proliferator-activated receptor δ in colorectal cancer

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) δ. In light of the recent finding that PPARδ is a target of β-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPARδ mRNA in matched normal and tumor samples revealed that expression of PPARδ, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPARδ is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPARδ mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPARδ and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI2) could serve as a ligand for PPARδ. In addition, the stable PGI2 analog, carbaprostacyclin, and a synthetic PPARδ agonist induced transactivation of endogenous PPARδ in human colon carcinoma cells. We conclude from these observations that PPARδ, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPARδ is transcriptionally responsive to PGI2. However, the functional consequence of PPARδ activation in colon carcinogenesis still needs to be determined.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

Top-down morphogenesis of colorectal tumors

One of the fundamental tenets of oncology is that tumors arise from stem cells. In the colon, stem cells are thought to reside at the base of crypts. In the early stages of tumorigenesis, however, dysplastic cells are routinely found at the luminal surface of the crypts whereas the cells at the bases of these same crypts appear morphologically normal. To understand this discrepancy, we evaluated the molecular characteristics of cells isolated from the bases and orifices of the same crypts in small colorectal adenomas. We found that the dysplastic cells at the tops of the crypts often exhibited genetic alterations of adenomatous polyposis coli (APC) and neoplasia-associated patterns of gene expression. In contrast, cells located at the base of these same crypts did not contain such alterations and were not clonally related to the contiguous transformed cells above them. These results imply that development of adenomatous polyps proceeds through a top-down mechanism. Genetically altered cells in the superficial portions of the mucosae spread laterally and downward to form new crypts that first connect to preexisting normal crypts and eventually replace them.

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.

APC mutations in colorectal tumors with mismatch repair deficiency.

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.

Expression of hyaluronidase by tumor cells induces angiogenesis in vivo.

Hyaluronic acid is a proteoglycan present in the extracellular matrix and is important for the maintenance of tissue architecture. Depolymerization of hyaluronic acid may facilitate tumor invasion. In addition, oligosaccharides of hyaluronic acid have been reported to induce angiogenesis. We report here that a hyaluronidase similar to the one on human sperm is expressed by metastatic human melanoma, colon carcinoma, and glioblastoma cell lines and by tumor biopsies from patients with colorectal carcinomas, but not by tissues from normal colon. Moreover, angiogenesis is induced by hyaluronidase+ tumor cells but not hyaluronidase- tumor cells and can be blocked by an inhibitor of hyaluronidase. Tumor cells thus use hyaluronidase as one of the "molecular saboteurs" to depolymerize hyaluronic acid to facilitate invasion. As a consequence, breakdown products of hyaluronic acid can further promote tumor establishment by inducing angiogenesis. Hyaluronidase on tumor cells may provide a target for anti-neoplastic drugs.

Apoptosis and APC in colorectal tumorigenesis.

Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death. In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice. While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery. Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth. Further evaluation demonstrated that this was due to the induction of cell death through apoptosis. These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts

Tagging of RNases, such as the ribotoxin α-sarcin, with the variable domains of antibodies directed to surface antigens that are selectively expressed on tumor cells endows cellular specificity to their cytotoxic action. A recombinant single-chain immunotoxin based on the ribotoxin α-sarcin (IMTXA33αS), produced in the generally regarded as safe (GRAS) yeast Pichia pastoris, has been recently described as a promising candidate for the treatment of colorectal cancer cells expressing the glycoprotein A33 (GPA33) antigen, due to its high specific and effective cytotoxic effect on in vitro assays against targeted cells. Here we report the in vivo antitumor effectiveness of this immunotoxin on nude mice bearing GPA33-positive human colon cancer xenografts. Two sets of independent assays were performed, including three experimental groups: control (PBS) and treatment with two different doses of immunotoxin (50 or 100 μg/ injection) (n = 8). Intraperitoneal administration of IMTXA33αS resulted in significant dose-dependent tumor growth inhibition. In addition, the remaining tumors excised from immunotoxin-treated mice showed absence of the GPA33 antigen and a clear inhibition of angiogenesis and proliferative capacity. No signs of immunotoxin-induced pathological changes were observed from specimens tissues.Overall these results show efficient and selective cytotoxic action on tumor xenografts, combined with the lack of severe side effects, suggesting that IMTXA33αS is a potential therapeutic agent against colorectal cancer.

Modulation of human V alpha 24(+)V beta 11(+) NKT cells by age, malignancy and conventional anticancer therapies

Immunotherapy strategies aimed at increasing human Valpha24(+)Vbeta11(+) natural killer T (NKT) cell numbers are currently a major focus. To provide further information towards the goal of NKT cell-based immunotherapy, we assessed the effects of age, cancer status and prior anticancer treatment on NKT cell numbers and their expansion capacity following alpha-galactosylceramide (alpha-GalCer) stimulation. The percentage and absolute number of peripheral blood NKT cells was assessed in 40 healthy donors and 109 solid cancer patients ( colorectal ( n = 33), breast ( n = 10), melanoma ( n = 17), lung ( n = 8), renal cell carcinoma ( n = 10), other cancers ( n = 31)). Responsiveness to alpha-GalCer stimulation was also assessed in 28 of the cancer patients and 37 of the healthy donors. Natural killer T cell numbers were significantly reduced in melanoma and breast cancer patients. While NKT numbers decreased with age in healthy donors, NKT cells were decreased in these cancer subgroups despite age and sex adjustments. Prior radiation treatment was shown to contribute to the observed reduction in melanoma patients. Although cancer patient NKT cells were significantly less responsive to alpha-GalCer stimulation, they remained capable of substantial expansion. Natural killer T cells are therefore modulated by age, malignancy and prior anticancer treatment; however, cancer patient NKT cells remain capable of responding to alpha-GalCer-based immenotherapies.

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with NISS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (P = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, NISS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).