Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK 20 RT-PCR. Detection rates are stage dependent, and survival analysis demonstrated statistically relevant data. From a clinical point of view, this finding is especially noteworthy for the group of well- and moderately-differentiated tumors.

SUMMARYThe interaction of CD28 with its ligands is important for T-cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7-1) and CD86 (B7-2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80-negative. CD80 expression is weakly induced after 6-8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4-6 hr in stimulated cells. Reverse transcription-polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80-mRNA. The mRNA of CD80 is induced after 4-6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti-CD80 and CTLA4Ig could partially inhibit antigenspecific (tuberculin) and polyclonal (anti-CD3) lymphoproliferation and interferon-g (IFN-g) secretion of T cells cocultured with autologous monocytes. IFN-g secretion was more sensitive to blocking costimulation than proliferation. The antibody BB-1 did not inhibit proliferation and cytokine secretion, nor did the anti-CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti-CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.

The dissemination of cancer cells is a prerequisite in the development of micrometastases and solid metastases. Our previous examinations of these cells were based on immunocytological staining of tumor-associated antigens and cytokeratins. We have now developed a highly sensitive and specific detection method based on a nested reverse-transcriptasepolymerase-chain reaction (RT-PCR) of The main problem after surgical treatment of patients with colorectal, gastric or pancreatic cancer is the frequent recurrence of disease. Routine diagnostic methods fail to detect circulating tumor cells, distant micrometastases or early metastases (Gusterson, 1991). The detectability of single tumor cells would be a matter of urgent necessity, not least because adjuvant therapy concepts have been proposed for elimination of these malignant cells (Moertel et al., 1990; Riethmuller et al., 1994). To date, the detection of disseminated single tumor cells in the bone marrow of patients with gastrointestinal cancer is performed mainly by immunocytological methods (Schlimok et al., 1987(Schlimok et al., , 1991Juhl et al., 1994). In order to establish a method with increased sensitivity, our group, like others (Traweek et al., 1993; Gerhard et al., 1994), chose a PCR-based detection procedure.The use of cytokeratin-20 as a promising marker for the detection of epithelial tumor cells in the bone marrow and the peripheral blood has been proposed by Burchill et al. (1995). CK-20 is a member of the epithelial sub-group of the multigene family of the intermediate filament proteins, the cytokeratins. The expression of CK-20 mRNA and protein is almost entirely confined to gastric and intestinal epithelium, urothelium and Merkel cells. Nearly all colorectal adenocarcinomas tested and many adenocarcinomas of the stomach and the pancreas revealed CK-20 mRNA and protein expression (Moll et al., . 1993. With the CK-20-specific nested RT-PCR we have established a sensitive and specific method for the detection of disseminated epithelial tumor cells. Bone-marrow samples of patients with malignant gastrointestinal diseases were examined for CK-20 mRNA expression. To ensure that this finding of CK-20 mRNA at the molecular level is dependent on the presence of carcinoma cells, we investigated bone-marrow samples of a control group from healthy donors and of patients with benign gastrointestinal diseases, hematological neoplasms and infectious diseases. MATERIAL AND METHODS Cell linesThe cell lines and culture conditions have been described (Kalthoff et al., 1991(Kalthoff et al., ,1993 (Dr. T. Okabe, Tokyo, Japan) from pancreatic carcinoma; MKN-7, MKN-28, Kato 111 (Dr. Hirai, Tokyo, Japan) from gastric carcinoma; SW1116 and HT29 from colorectal carcinoma (Dr. W. Dippold, Mainz, Germany); Huh7 (Dr. P. Galle, Heidelberg, Germany) and MZ-1 (Dr. W. Dippold) were from liver carcinoma. Bone-marrow samplesHeparinized bone-marrow aspirates ( 5 ml) were obtained from patients with colorectal carcinoma (n = 57) and pancreatic carcinoma (n = 11). As a control,...

Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30-60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.