Effects of ectopic overexpression of p21(WAF1/CIP1) on aneuploidy and the malignant phenotype of human brain tumor cells

p21WAF1/CIP1 is a downstream effector of the p53 tumor suppressor gene and a universal cyclin-dependent kinase (CDK) inhibitor. To determine the ability of p21WAF1/CIP1 to function as a tumor suppressor, we constructed a replication-defective adenovirus vector containing p21WAF1/CIP1 (Adp21WAF1/CIP1) to effect ectopic overexpression in a p53-defective human astrocytoma cell line, U-373MG. We observed a marked decrease in CDC2 and CDK2 kinase activity associated with a corresponding decrease in the amount of CDC2 but not CDK2 protein; a decreased growth potential of Adp21WAF1/CIP1-infected cells demonstrated by diminished [3H]thymidine incorporation, increased cell doubling time and G1-arrested cell cycle; an association between Adp21WAF1/CIP1-infected cells and inhibition of aneuploid cell accumulation; and an alteration of the malignant phenotype of cells was evidenced by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both peripheral and intracerebral xenograft models, including the prevention of tumor formation Adp21WAF1/CIP1 infection 2 days post tumor cell implantation. Adp21WAF1/CIP1. Adp21WAF1/CIP1 appears to be a strong candidate for gene therapy studies based on these studies indicating that Adp21WAF1/CIP1 inhibits proliferation, tumorigenicity and aneuploidy in human brain tumor cells.     

A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and loss of cyclin-Cdk activity in human breast cancer cells

In order to elucidate the biochemical mechanisms by which the universal cyclin kinase inhibitor p27Kip1 regulates cell cycle progression in human breast cancer cells, a recombinant adenovirus expressing human p27 was constructed (Adp27). Upon infection of human breast cancer cells MDA-MB-231 and MCF-7 with Adp27, a high level of p27 expression was observed, and this resulted in a marked decrease in the proportion of cells in S-phase. In multiple cell lines, comparison of the cytotoxicity of Adp27 with another adenovirus vector expressing the related universal cyclin kinase inhibitor WAF1/Cip1 (AdWAF1), showed Adp27 to be markedly more (up to 56-fold) toxic than AdWAF1. DNA histograms showed Adp27 to cause a G1/S arrest at lower viral doses than AdWAF1. Analysis of cyclin dependent kinase activity following Adp27 infections showed decreased Cdk2 and cyclin B1-Cdc2 activity at lower viral doses when compared with AdWAF1. Adp27 is therefore potentially useful for studies of growth regulation and for gene therapy when growth inhibition is desired.     

Wild-type p53 protein potentiates cytotoxicity of therapeutic agents in human colon cancer cells

Wild-type p53 is induced by DNA damage. In different cell types, this induction is suggested either to facilitate DNA repair by inducing a cell cycle pause or to potentiate cell death via apoptosis. Wild-type p53 in different cell types has similarly been associated with either enhancement of or increased resistance to the cytotoxicity of many cancer therapeutic agents. We have constructed a colorectal cancer cell line bearing, in addition to endogenous mutant p53 alleles, an exogenous wild-type p53 allele that is under the regulatable control of the lac repressor. Induction of wild-type p53 by isopropyl-beta-thiogalactopyranoside in these cells induces a reversible growth arrest but does not induce cell death. However, we find that the induction of wild-type p53 powerfully potentiates the cytotoxicity of both irradiation and 5-fluorouracil, two agents that are used clinically in the treatment of colorectal cancer. We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer. These findings suggest that the common loss of wild-type p53 in many colorectal cancers may play a role in the clinical resistance of these tumors to anticancer agents. Although some cancer cells may not be directly killed by p53 gene therapy, our findings suggest that genetic alteration of some cancers to induce wild-type p53 may increase their sensitivity to cytotoxic gene therapy.     

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135

The last two decades have led to a greater understanding of the genetic basis of human malignancy. Although numerous genetic alterations have been detected in cancer, activation of oncogenes and inactivation of cell cycle regulators (e.g., tumor suppressor genes) are now known to play a critical role in the progression of the disease. Therapeutic strategies based on specific molecular alterations in cancer include reintroduction of wild-type tumor suppressor function to cells lacking the gene. p53 gene therapy provides an attractive strategy to test the potential clinical feasibility of this approach. Alterations in p53 function are present in approximately half of all malignancies, and expression of wild-type p53 can result in apoptosis in human tumor cells. This review summarizes current investigations with p53 gene therapy, highlighting the preclinical efforts with adenoviral, retroviral, and lipid-based gene delivery systems. A comprehensive review of the various clinical targets suggested for p53 gene therapy is presented together with challenges and prospects for future clinical investigation.     

Genetic alterations and molecular mechanisms underlying colorectal tumorigenesis

Tumor cells are cells that have acquired damage to genes that directly regulate their cell cycles. In the multistep process leading to colorectal carcinoma, the adenoma-carcinoma sequence is characterized by progressive accumulation of genetic abnormalities (K-ras oncogene mutation, allelic deletion on chromosome 5q, 18q, 17p). In a hereditary non-polyposis syndrome (Lynch syndrome II) and in about a quarter of the cases of sporadic colorectal cancer there is a DNA micro-instability which contributes to the acquisition of mutations that cause loss of tumor-suppressor function. The p53 tumor-suppressor gene is the most frequently mutant gene in human cancer. In colorectal cancer cells missense p53 mutations and allelic deletion on chromosomal locus 17p13.1 are found with very high frequency. One of biological roles of p53 gene is to ensure that, in response to genotoxic damage, cells arrest in G1 and attempt to repair their DNA before it is replicated. In addition, p53 is required for apoptosis in response to severe DNA damage, included the damage induced by chemotherapeutics drugs and ionizing radiation. The loss of p53 function results in genomic instability and has been implicated in the evolution of normal cells into cancer cells.     

Risk and preventive factors for cot death in The Netherlands, a low-incidence country

BACKGROUND: The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. METHODS: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. RESULTS: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. CONCLUSIONS: Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.     

Expressions of cell cycle regulators in human colorectal cancer cell lines

To study the altered mechanisms of cell cycle regulation in colorectal cancer, the expressions of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, p53 and retinoblastoma (Rb) protein were analyzed by western blotting in a series of human colorectal cancer cell lines. The colorectal cancer cell lines exhibited various expression patterns of cell cycle regulators, which may reflect differences in the biological characteristics of cancer cells and in the genetic backgrounds of carcinogenesis. A correlation was found between p53 gene alteration and p21 expression, suggesting that p53 gene mutation usually suppresses p21 expression, though p21 expression could be induced via both a p53-dependent and a p53-independent pathway in colorectal cancer. None of the cell lines studied expressed p16 protein, suggesting that inactivation of p16 may be a common alteration in colorectal cancer. Moreover, all the D-type cyclins, especially D2 and D3, were expressed at a high level in most of the cell lines. Loss of p16 expression and increased expression of D-type cyclins promote CDK-mediated Rb phosphorylation. All of the colorectal cancer cell lines studied herein expressed Rb protein, but the growth-suppressive properties of Rb may be inactivated by the loss of p16 expression and increased expressions of D-type cyclins. In view of the pivotal role of Rb in cell cycle regulation, loss of p16 expression and overexpression of D-type cyclins may be critical alterations in colorectal cancer.     

Colour-coded duplex sonography of preocclusive carotid stenoses

Gene replacement therapy is potentially a very powerful tool, targeting specific molecular mediators of cancer development and progression. p21WAF1 (p21) is a cyclin-dependent kinase inhibitor that is induced by p53 upon DNA damage or p53 overexpression, resulting in cell cycle arrest at the G1 checkpoint and inhibition of further cell proliferation. Using a replication-deficient recombinant adenovirus (AdV) ((rAd)-p21) as a p21 gene delivery system, we have evaluated the effect of p21 introduction in lung cancer cells in vitro as well as in vivo. In in vitro experiments, two human non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and NCI-H322, showed dose-dependent p21 induction and concomitant cell growth inhibition following rAd/p21 infection. Flow cytometric analysis of the cell cycle revealed a significant increase in the percentage of NCI-H460 cells in G0/G1 following rAd/p21 infection compared with untreated cells, suggesting that p21-induced growth inhibition was due to G0/G1 arrest. We also tested the therapeutic efficacy of rAd/p21 in an in vivo human NSCLC model in SCID mice. Tumor-bearing mice were established by subcutaneous injections of NCI-H460 cells and treated by repeated intratumoral injections of rAd/p21. Intratumoral delivery of rAd/p21 significantly suppressed tumor growth and prolonged survival in rAd/p21-treated mice. Our in vitro and in vivo results provide strong preliminary evidence for the growth inhibition of NSCLC by rAd/p21. Collectively, these results justify further studies to evaluate the efficacy of rAd/p21 for gene therapy in human lung cancer.     

The levels of integrin alpha v beta 5 may predict the susceptibility to adenovirus-mediated gene transfer in human lung cancer cells

Adenovirus mediated transfer of growth-inhibiting molecules, such as p53 shows promise as an effective method of suppressing the growth of cancer cells. As the basis for in vivo studies, we examined transfection efficiency using 15 human lung cancer cell lines that differ in their endogenous p53 status. When infected with an adenovirus expressing bacterial beta-galactosidase, the different cell lines showed different levels of beta-galactosidase activity. We found a correlation between the level of integrin alpha v beta 5, which is thought to be an adherence receptor for adenoviruses, and the expression level of the transferred gene, suggesting that gene expression is largely dependent on the infection efficiency. Growth inhibition was induced in all cell lines tested following infection with an adenovirus containing p53, regardless of the genetic status of their endogenous p53 provided a sufficient amount of p53 protein was expressed. Our results (1) confirm that the examination of the susceptibility of target cancer cells to an adenovirus is important when considering performing adenovirus-mediated gene transfer and for evaluating its therapeutic effects; and (2) suggest that the quantification of integrin alpha v beta 5 may be a good way of predicting the susceptibility of cells to adenoviral vectors.     

p53 gene therapy in vivo of herpatocellular and liver metastatic colorectal cancer

Tumor suppressor genes such as p53 contribute to the oncogenic process via loss-of-function mechanisms such as genetic mutation or complex formation with other cellular or viral proteins. p53 is mutated in approximately 50% of human tumors and has an important role in the genesis or progression of both colorectal and hepatocellular cancers. Colorectal cancer is leading cause of cancer mortality in the United States, whereas hepatocellular cancer is the leading worldwide cause of cancer death; the liver is a primary site of morbidity in both diseases. Because systemic tumor suppressor gene therapy is currently not feasible, we have chosen to develop a regional form of such therapy directed at primary or metastatic liver neoplasms. Gene replacement therapy with p53 is a promising new strategy to treat advanced human cancers.     

Localization by immunoelectron microscopy of Schistosoma mansoni antigens in the glomerulus of the hamster (Mesocricetus auratus) kidney

Gene therapy has the potential to provide cancer treatments based on novel mechanisms of action with potentially low toxicities. This therapy may provide more effective control of loco-regional recurrence in diseases such as non-small cell lung cancer (NSCLC), as well as systemic control of micrometastases. Despite current limitations, retroviral and adenoviral vectors can in certain circumstances provide an effective means of delivering therapeutic genes to tumour cells. Although multiple genes are involved in the process of carcinogenesis, mutations of the p53 gene are the most frequent abnormality identified in human tumours. Pre-clinical studies both in vitro and in vivo have shown that restoration of p53 function can induce apoptosis in cancer cells. Phase I clinical trials now show that p53 gene replacement therapy is feasible and safe using both retroviral and adenoviral vectors, and that it induces tumour regression in patients with advanced NSCLC and recurrent head and neck cancer. Other pre-clinical studies indicate that gene therapy may have useful synergy with cytotoxic and radiation therapy. This paper describes the different gene therapy strategies under investigation and the pre-clinical data that provides a rationale for the gene replacement approach, reviews clinical trial data and presents novel ideas for improving current vectors and gene delivery to tumours.     

Population dynamics of the deer mouse (Peromyscus maniculatus) and Sin Nombre virus, California Channel Islands

Alteration of the wild-type (wt) p53 gene by mutation, deletion or re-arrangement is a major factor in the development of human colon cancer. Recent studies have demonstrated that p53 might be an essential component of the apoptotic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We examined the anti-tumor effects of adenovirus-mediated wt-p53 gene transfer in combination with a chemotherapeutic drug on the human colon cancer cell line WiDr, which is homozygous for a mutation in the p53 gene. Treatment with the chemotherapeutic drug cisplatin following infection with a replication-deficient, recombinant adenoviral vector expressing wt-p53 (termed AdCMVp53) significantly suppressed the growth of WiDr cells compared to single treatments alone. To evaluate the in vivo efficacy of AdCMVp53 and cisplatin given sequentially, WiDr cells were inoculated s.c. in nu/nu mice. After 3 days, AdCMVp53 was injected s.c. into the area where tumor cells were implanted, followed by i.p. administration of cisplatin. Analysis of initial growth inhibition at 21 days demonstrated a profound therapeutic cooperativity, though administration of either AdCMVp53 or cisplatin alone was followed only by a slowing of growth. Our results suggest that gene therapy using wt-p53-expressing adenovirus in combination with a chemotherapeutic DNA-damaging drug could be a useful strategy for treating human colon cancer.     

Molecular events including p53 and k-ras alterations in the in vitro progression of a human colorectal adenoma cell line to an adenocarcinoma

The aim of the current study was to identify genetic abnormalities in human colorectal adenoma and carcinoma derived cell lines, and to determine whether the genetic changes which occur in vitro are relevant to the in vivo situation. Loss of 1p(33-35) region was shown to be the most common chromosome 1 abnormality and loss of heterozygosity (LOH) of the DCC gene and/or adjacent sequences was detected in all adenoma derived cells as well as the carcinoma cell lines. The level of p53 protein was also investigated as increased cellular p53 protein had previously been associated with mutation of the p53 gene. A further aim was to investigate genetic changes in our in vitro model of tumour progression, where the adenoma derived PC/AA cell line has previously been converted in vitro to two distinct tumorigenic phenotypes, producing either an adenocarcinoma or a mucinous carcinoma in athymic nude mice. Progression to the adenocarcinoma phenotype was shown to involve a specific chromosome 1 rearrangement, loss of both normal copies of chromosome 18 (although DCC gene sequences were retained), loss of the remaining wild type allele of k-ras resulting in homozygosity for the k-ras codon 12 mutation and increased cellular p53 protein as detected by SDS-PAGE Western blotting. The increase in p53 protein was shown not to be due to the acquisition of a mutation in the p53 gene. Interestingly, progression of the adenoma derived PC/AA cell line to the mucinous malignant phenotype did not involve any of these molecular rearrangements, suggesting that different genetically distinct pathways are involved in colorectal carcinogenesis. These studies show that the genetic changes in our in vitro model of human colorectal tumour progression are similar to those observed in in vivo studies.     

High-resolution magnetic resonance imaging: three-dimensional trabecular bone architecture and biomechanical properties

The development of vectors that are capable of efficient gene delivery is crucial to the success of gene therapy. We have developed both recombinant viral and nonviral vectors with the goal of correcting genetic abnormalities in cancer cells that are responsible for malignant transformation. Infection of cancer cells by recombinant adenovirus (Adv) indicates that the level of transduction is variable and dependent on the virus-to-cell ratio. Infection of cells with Adv/p53 resulted in levels of tumor suppressor p53 gene expression that could mediate tumor cell growth suppression and apoptosis, both in vitro and in vivo. The treatment of cancer cells with cisplatin prior to Adv transduction resulted in a higher level of therapeutic gene expression. Epidermal growth factor (EGF)/DNA complexes targeted to cancer cells overexpressing the EGF receptor resulted in efficient transduction of several lung cancer cell lines in vitro. As a result, these vectors provide improved methods with which to treat cancer in the clinical setting with gene therapy.     

Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations

Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.     

Effective treatment of early endobronchial cancer with regional administration of liposome-p53 complexes

BACKGROUND: Lung cancer originates in a diffusely damaged bronchial epithelium as a result of sequential and cumulative genetic alterations. We investigated the feasibility of in vivo gene replacement in endobronchial precancerous and cancerous cells by a regionally administered nonviral delivery system. METHODS: After evaluating the in vitro transfection efficiency and cytotoxicity of a variety of cationic liposome-p53 formulations, a specific formulation, DP3-p53, was selected for further in vitro and in vivo evaluation. The ability of DP3-p53 to introduce the p53 gene in the normal bronchial epithelium was studied in transgenic mice that lack the p53 gene. The therapeutic effect of DP3-p53 administered intratracheally was studied in two nude mouse models of endobronchial human lung cancer by use of H358 (p53-null) and H322 (p53-mutant) cells. RESULTS: DP3-p53 was able to effectively introduce and express the p53 gene and induce G1 arrest and apoptosis in H358 cells in vitro and to introduce and transcribe the p53 gene in the bronchial epithelium of transgenic mice that lack the p53 gene in vivo. In therapeutic experiments using groups of four or five mice each, administration of five intratracheal doses of DP3-p53 (2 microg or 8 microg DNA per dose) on days 4, 8, 12, 16, and 20 after intratracheal tumor inoculation significantly inhibited lung tumor formation and prolonged by approximately twofold the survival of mice bearing H358 or H322 endobronchial tumor cells in contrast to the survival among untreated mice and mice treated with the DP3-empty vector (P = .007 [two-sided logrank test] for mice bearing H358 cells and P = .008 [two-sided logrank test] for those bearing H322 cells). CONCLUSIONS/IMPLICATIONS: Liposome-based p53 delivery through the airways is a potentially effective strategy for the treatment of early endobronchial cancer. These results have important implications for the gene therapy and prevention of human lung cancer.     

A newly developed adenovirus-mediated transfer of a wild-type p53 gene increases sensitivity to cis-diamminedichloroplatinum (II) in p53-deleted ovarian cancer cells

A new recombinant adenovirus carrying a wild-type p53 gene (AxCAp53) was developed and the combination effect of p53 gene transfer and cis-diamminedichloroplatinum (II) (CDDP) was examined in an ovarian cancer cell line, SK-OV-3, with deletion of the p53 gene. AxCAp53 showed a high efficiency of gene transduction and increased sensitivity to CDDP in the SK-OV-3 cells. It was found that the sensitivity of the cells to CDDP correlated with the amount of infectious units of virus per cell of AxCAp53 which correlated with p53 protein expression. The results suggest that the combination of CDDP and AxCAp53 may be a potential strategy for the therapy of CDDP-resistant ovarian cancer.     

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.     

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

BACKGROUND/AIMS: Mutations in p53, a tumor suppressor gene located on chromosome 17p, are the most frequent genetic alterations found in human cancers. Increased intracellular concentration of p53, which is frequently but not systematically related to p53 mutation, has been proposed to be associated with poor prognosis in some tumor types. In colorectal cancer, this significance is still a matter of debate. To directly investigate the relationship between prognosis and p53 mutation, this study screened a series of 85 colorectal carcinomas for mutations in exons 5-8 of this gene. METHODS: Polymerase chain reaction-amplified products from tumor DNA were analyzed by denaturing gradient gel electrophoresis and direct DNA sequencing. RESULTS: Forty-four tumors were found to be mutated (52%). A strong correlation between the presence of a mutation and short survival was observed (P = 0.003). When tumors were classified according to their histological stage, a multivariate Cox model analysis showed that p53 mutation, rather than 17p allelic loss (previously proposed to convey prognostic information), was retained as the only independent prognostic factor (relative risk, 2.25; 95% confidence interval, 1.06-4.80; P < 0.029). CONCLUSIONS: Combined with staging, direct monitoring of p53 mutation improves prognostic accuracy for colorectal cancer.