Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied as an alternative to ex-vivo bromodeoxyuridine (BrdU) immunohistochemistry for assessment of human colonic cell proliferation. From 16 subjects without colonic disease biopsy specimens were collected from five different sites along the colorectum and processed for BrdU Materials and methods BIOPSY SPECIMENSBiopsy specimens of endoscopically normal colonic mucosa were taken at five different sites from proximal to distal in the colorectum (ascending, transverse, and descending colon, sigmoid, and rectum) of 16 subjects who had had diagnostic colonoscopy because of abdominal complaints and who were found not to have any organic colonic disorders (seven men, nine women, mean age 49 years, range 17 to 76). All specimens were taken by one endoscopist (LGJBE) and completely processed by the same technician (AP-H). Specimens were immediately placed in RPMI 1640 tissue culture medium containing 10% fetal calf serum, 20 [1M 5-fluoro-uracil, 20 FM bromodeoxyuridine (Serva, Heidelberg, Germany), and 0 9% hydrogenperoxide during one hour at 37°C.The composition of the tissue culture medium was based on a 3 x 3 experiment in which optimal conditions for ex vivo labelling of mouse and human tissue with BrdU were determined. Intestinal specimens were incubated in RPMI 1640 medium containing 10% fetal calf serum and concentrations of 1, 2 or 3x 10-) M BrdU and 0 5, 1 or 2 10-5) M 5-fluorouracil. We found that the addition of hydrogen peroxide seemed to improve the label uptake, therefore in this experiment hydrogen peroxide 30% was added to final concentrations of 0-3, 0-9, and 1-8% respectively. Specimens were immersion fixed in 70% ethanol for at least 24 hours. Best results with regard to immunoreactivity and morphology, were obtained by incubation with RPMI 1640 medium containing 10% fetal calf serum, 2x 10-) M 5-fluoro-uracil, and 0-9% hydrogen peroxide.As maximum incorporation of BrdU can be expected after depletion of the endogenous pool 530 on 12 May 2018 by guest. Protected by copyright.

Human cancer is characterized by genetic and epigenetic alterations. In this study we provide evidence for the interruption of Ras signaling in sporadic colorectal cancer (CRC) by either genetic activation of the K-ras oncogene or epigenetic silencing of the putative tumor suppressor gene RASSF1A. Paraffin embedded tumor tissue samples from 222 sporadic CRC patients were analysed for K-ras codon 12 and codon 13 activating mutations and RASSF1A promoter hypermethylation. Overall, K-ras mutations were observed in 87 of 222 (39%) and RASSF1A methylation was observed in 45 of 222 (20%) of CRCs. Mutation of K-ras alone was detected in 76 of 222 (34%) CRCs. RASSF1A promoter methylation with wild-type K-ras was observed in 34 of 222 (15%) CRCs. In 101 of 222 (46%) CRCs neither K-ras mutations nor RASSF1A methylation was observed and 11 of 222 (5%) CRCs showed both K-ras mutations and RASSF1A methylation. These data show that the majority of the studied CRCs with K-ras mutations lack RASSF1A promoter methylation, an event which occurs predominantly in K-ras wild-type CRCs (P=0.023, Chi-square test).

In order to assess the effect of the no-touch isolation technique, in the treatment of large bowel cancers, on the site of first recurrence and disease-free and overall survival, 236 patients were prospectively and randomly assigned to either the no-touch isolation technique (117 patients) or to a conventional resection technique (119 patients). No patient with distant metastases or unresectable disease entered the study. The two treatment groups were comparable with regard to patient characteristics. Pre- and postoperative complications (including mortality within 30 days) were similar in both groups. After a complete follow-up of 5 years, a tendency for reduction in the number of, and time to, occurrences of liver metastases was seen in the no-touch isolation group (P = 0.14). This effect was most obvious in the sigmoid colon with angio-invasive growth. Overall (P = 0.42) and corrected (P = 0.25) survival did not differ significantly among the treatment groups although in every analysis the survival data of the no-touch isolation group were superior. The data do suggest a limited benefit of the no-touch isolation technique. This observation is important since the morbidity and mortality of surgery were equal in both groups.

The clinical, laboratory, and pathologic data of 310 patients who had curative resections were prospectively collected and analyzed in a multiple stepwise regression model. Although several factors (i.e., venous invasion) were of importance in univariate analysis, the following conclusions reflect the outcome and relative importance of the regression analysis only. Blood loss as an initial symptom and duration of symptoms were associated with a better prognosis. Location of the primary tumor, age, and sex did not appear to have prognostic value. Observations during operation such as palpable lymph nodes, fixity to adjacent organs, and tumor spill were related to a diminished tumor-free survival. Laboratory data (hemoglobin, leukocytes, ESR, GGTP, SGOT, SGPT, LDH, total protein, CEA) were tested for their potential prognostic values. Only a preoperative low protein level or an elevated CEA level were associated with an increased risk of death due to recurrent tumor. The histopathologic features (stage and grade), with the exception of venous invasion, were of relative importance in the determination of prognosis. The aforementioned variables can be included in a prognostic index on the base of which high-risk groups suitable for adjuvant studies can be identified.

Mutations in the adenomatous polyposis coli (APC) gene are linked to the dysplastic transformation of colorectal polyps and represent an early step in the development of colorectal tumors. Ninety-four percent of all mutations result in the expression of a truncated APC protein lacking the C-terminal region. The C-terminal region of the APC protein may have a tumor suppressor function as its absence appears to be linked to the development of dysplastic lesions. Recently, we discovered and characterized a protein called RP1 which binds specifically to the C-terminal region of the APC protein. We show now that RP1 and the other known members of the EB/RP family (EB1 and RP3) also bind directly to tubulin, both in vitro and in vivo. Immunohistochemical analyses reveal a distinct staining pattern during interphase as well as an association of RP1/EB1 with mitotic microtubule structures. The previously described puncta of the APC protein at the leading edge of membrane protrusions contact microtubule fibers that contain RP1 or EB1. Int.