SUMMARY:Several techniques to determine apoptotic frequencies in tumors have been described. In this study, we report that biochemical detection of enzymatic caspase-3 activity is a simple and quantitative technique to measure apoptosis in colorectal tumor cells. The relevance of the level of apoptosis in colorectal cancer for the clinical course remains unclear. Therefore, we studied the correlation between caspase-3 activity and prognosis of the disease in relation to different factors known to be involved in apoptosis induction. High caspase-3 activity significantly correlated with a higher risk of recurrence and was preferentially found in tumors of the right side of the colon. No correlation was detected between high caspase-3 activity and altered protein expression of p53, ␤-catenin, or proteins of mismatched repair genes. This indicates that high caspase-3 activity has no evident correlation with the genetic Wnt-signaling or the mismatch repair mutational pathways. The caspase-3 activity significantly correlated with CD57 ϩ tumor infiltrating cells. Therefore, high caspase-3 activity in right-sided tumors might be induced by a specific lymphocytic reaction. (Lab Invest 2001, 81:681-688).A poptosis is an essential biologic process. As well as having a role in controlling cell number during early development, apoptosis is important for the removal of infected or genetically altered cells (Duke et al, 1996). Defects in the apoptotic mechanism are often found in neoplastic growth (Duke et al 1996;Green and Martin, 1995;Tompson, 1995), which is also the case in colorectal cancer (Evertsson et al, 1999;Gryfe et al, 1997;Tsujitani et al, 1996). Development of colorectal cancer proceeds through a series of genetic alterations that result in the activation of oncogenes and loss of tumor suppressor genes (Gryfe et al 1997;Rafter and Glinghammar, 1998;Ilyas et al, 1999). Mutations in genes known to be involved in cell cycle regulation, such as APC, p53, ␤-catenin, deleted in colorectal cancer (DCC), and K-ras, have been reported in colorectal cancer (reviewed in Gryfe et al, 1997). Furthermore, mutated genes involved in DNA mismatch repair can contribute to tumor growth. Alterations in these mismatch repair genes will lead to a microsatellite instability phenotype (MSI) (reviewed in Lothe, 1997). During tumorigenesis one of the target genes that is preferentially inactivated due to MSI is transforming growth factor ␤ receptor II (TGF␤RII) (Markowitz et al, 1995;Akiyama et al, 1996). Inactivation of TGF␤RII on cells down-regulates the suppressive function of TGF␤ on cell proliferation and in that way also results in disturbance of growth control (DeVisser and Kast, 1999). Most of the hereditary nonpolyposis colorectal tumors (HNPCC) exhibit MSI, whereas in sporadic colorectal cancers only 3% to 10% show this phenotype. This low rate of MSI in sporadic colorectal cancers seems to be caused by somatic inactivation of hMLH1 (Lothe, 1997).As well as the occurrence of apoptosis in cells caused by intrinsic factors, apopto...

Peripheral blood natural killer (NK) cells are usually defined as a homogeneous cell population. However, NK cells show heterogeneous expression of a diversity of cell surface molecules, which might reflect the diversity of NK-cell functions. Therefore, a more specific phenotypic definition of NK cells is necessary. In this study, we made an inventory of phenotypic subsets that are present within the peripheral blood NK-cell population of healthy donors based on differential expression of nine cell-surface markers. Using threecolour flow cytometric analysis we were able to define at least 48 different CD56 1 NK-cell subsets within the peripheral blood. This phenotypic heterogeneity appeared to be stable among healthy individuals, and was also steady within CD56 dim and CD56 bright NK populations, indicating a possible role for these subsets in NK-cell function or differentiation.

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 8 plaque-forming units of the hIL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors

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