Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements.

[Tumor cell dissemination in bone marrow and peritoneal cavity. An immunocytochemical study of patients with stomach or colorectal carcinoma]. / Tumorzelldissemination in das knochenmark und in die peritonealhoehle. Eine immunzytochemische untersuchung an patienten mit einem magen; oder kolerektalen karzinom.

The tumor spread and the radicality of surgical resection are the most important facts in a patient's prognosis. In spite of curative tumor resection many patients die from metastases or local tumor recurrence. One possible reason is early dissemination of tumor cells which cannot be detected with clinical methods of examination. For this reason the aim of our study was to examine both bone marrow and peritoneal lavage for disseminated tumor cells with an immunocytochemical technique in patients with a gastrointestinal carcinoma. We also wanted to find out whether there was any correlation between the incidence of tumor cell detection and the TNM classification, staging and tumor grading and whether disseminated tumor cells have any prognostic significance. Our study included 54 patients who underwent surgery in our clinic for a carcinoma of the stomach (20 patients) or the colorectum (34 patients) from November 1993 to December 1994. At the beginning of the operation bone marrow had been taken from the iliac spine, and the abdomen was irrigated with 1000 ml saline solution immediately after laparotomy or laparoscopy. After cell separation with Ficoll density centrifugation 5 x 10(5) cells were applied per slide by a cytospin technique. For detection of the tumor cells we used the APAAP technique and the following monoclonal antibodies: KL1, CK2, anti-CEA, 17-1A (bone marrow) and Ber-EP4, B72.3, anti-CEA and 17-1A (peritoneal lavage). Altogether 77% of all patients had tumor cells in the bone marrow and 69% in peritoneal lavage fluid. It was possible to detect tumor cells in bone marrow (67%) and peritoneal lavage fluid (25%) even of patients with T1 tumors. The percentage increased with depth of wall infiltration. There was a marked difference in bone marrow aspirates between patients with lymph-node-negative tumors (N0) and those with lymph-node-positive tumors (N+): 65% had tumor cells in N0 and 85% in N+ stages. This trend was also seen in patients with (M1) and without (M0) metastases, in both bone marrow aspirates and peritoneal lavage fluid. In bone marrow there was a good correlation of tumor cells with staging, but in peritoneal lavage fluid this was not so. Finally, we detected tumor cells more often in bone marrow and peritoneal lavage fluid of patients with poorly differentiated tumors (G3) or diffuse Lauren type than in patients with moderately differentiated tumors (G2) or intestinal Lauren type. After a median follow-up period of 12.5 months patients with disseminated tumor cells had a lower survival rate than patients without tumor cells.