c-Ha-ras-I oncogene-induced differentiation and natural killer cell resistance in a human colorectal carcinoma cell line.

Natural killer (NK) activity is primarily a peripheral blood function of a lymphocyte population capable of spontaneous lysis of many transformed and metastatic targets. However, NK-susceptible targets tend to be relatively poorly differentiated. We have previously shown that poorly differentiated human colorectal carcinoma are lysed by NK cells. Well-differentiated and chemically differentiated colorectal carcinomas are insensitive to NK lysis. The present study demonstrates that transfection of the c-Ha-ras-I oncogene into a poorly differentiated colorectal carcinoma cell line also renders it NK resistant. This resistance is accompanied by a more differentiated colorectal carcinoma phenotype. Two ras-transfected lines (Clone-A-5 and Clone-A-4) showed a 30-66% decrease in susceptibility to NK lysis as compared to the parental line in standard cytotoxicity assays. The resistance of these transfectants was strictly dependent on expression of the activated p21, the H-ras protein product. Studies to assess the integrity of the initial binding step in NK lysis showed a significant decrease in the ability of these transfectants to form conjugates with fresh NK cells. It is likely that transfection with c-Ha-ras-I has selectively modulated critical NK target recognition structures.

LU103793 (NSC D-669356): a synthetic peptide that interacts with microtubules and inhibits mitosis.

LU103793 (NSC D-669356) is a new synthetic derivative of Dolastatin 15, an antiproliferative compound which was isolated from the mollusk Dolabella auricularia. Like Dolastatin 15, LU103793 is highly cytotoxic in vitro (IC50 = 0.1 nM). To investigate the mechanism of action of LU103793, we used a combination of biochemical and cellular methods. Turbidity assays with bovine brain microtubules demonstrated that LU103793 inhibits microtubule polymerization in a concentration-dependent manner (IC50 = 7 microM). Treatment with this compound also induced depolymerization of preassembled microtubules. Cell cycle analysis of tumor cell lines treated with LU103793 indicated a block in the G2-M phase. At the cellular level, it induced depolymerization of microtubules in interphase cells and development of abnormal spindles and chromosome distribution in mitotic cells. Although these effects are very similar to the cellular alterations caused by vinblastine, LU103793 does not inhibit vinblastine binding to unpolymerized tubulin in vitro. Our results suggest that LU103793 exerts its cytotoxic activity primarily through disruption of microtubule organization.

Natural killer sensitivity of colorectal carcinoma targets. Correlation with degree of differentiation.

Lymphocytes with natural killer (NK) ability recognize a wide range of target cells; however, the mediators of specific target recognition are still largely unknown. Despite the diversity of the NK target repertoire, it is typified by cells of relatively immature phenotype. The purpose of this study was to determine if colorectal carcinoma (CRC) targets were sensitive to spontaneous lysis by fresh human NK effector cells and to observe the role of CRC differentiation in this process. Results demonstrated that poorly differentiated CRC targets were susceptible to NK-mediated lysis whereas CRC targets with more differentiated structure were NK resistant. In addition, chemical induction of a more mature CRC phenotype confers NK resistance on a previously NK-susceptible target. This maturation-related modulation of NK sensitivity can provide a model with which to further study interaction between NK cells and CRC targets.

Inhibition of pluripotent hematopoietic stem cells of bone marrow by large granular lymphocytes.

Previous studies suggested that natural killer (NK) cells are involved in the regulation of the growth and differentiation of pluripotent hematopoietic stem cells. To establish whether the effector cells responsible for the in vivo resistance to bone marrow (BM) transplants and the in vitro inhibition of colony-forming units (CFU) may represent identical or overlapping populations, we used a rat system for syngeneic BM transplantation, with and without the transfer of large numbers of peripheral blood large granular lymphocytes (LGLs). BM reconstitution was measured by the in vivo formation of syngeneic CFU in the spleen (CFU-s). Because of the very low frequency of CFU-s in normal rat BM, we fractionated BM cells in Percoll density gradients, which provided a 2- to 5-fold enrichment in CFU-s in the lower-density fractions. Although these fractions contained less than 10% of the total cells, they contained greater than 75% of the CFU-s and allowed for the transfer of significantly fewer donor cells. At the time of BM transplantation, radiation-resistant asialoganglioside GM1-positive LGLs, with high NK activity, accounted for a significant percentage of the lymphoid cells in the irradiated recipient. The in vivo regulatory role of these cells on engraftment was demonstrated by their depletion (by i.v. injection of small amounts of anti-asialo-GM1 antiserum before BM transplantation), which resulted in a significant increase in the number of CFU-s. Conversely, a 50% inhibition in CFU-s was found when CFU-s-enriched BM fractions were preincubated in vitro with LGLs. Additional experiments, involving selective in vivo depletion of NK cells followed by LGL repopulation, directly demonstrated the involvement of LGLs in the regulation and growth of syngeneic pluripotent hematopoietic stem cells. Our results further support the hypothesis that LGLs are involved directly or via humoral factors in the homeostasis and regulation of hematopoietic stem cell growth and differentiation.

In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats.

The present study was performed to further evaluate the possible in vivo involvement of natural killer (NK) cells in host resistance against tumors. Selective depression of NK activity in Wistar Furth rats was induced by i.p. or i.v. injection of rabbit anti-asialo GM1. This antiserum has previously been shown to produce a decrease in NK activity and a parallel increase in tumor growth in mice. In the present study, rats treated with this antibody showed a parallel decrease in NK activity and in the frequency of large granular lymphocytes (LGL) in the spleen and peripheral blood, indicating that the antiserum-induced depression of NK activity in these sites was probably caused by an elimination of most effector cells. To further determine the possible role of rat LGL in tumor rejection in vivo, we studied LGL involvement in the rapid clearance of radiolabeled tumor cells from the lungs, an assay previously shown to correlate well with in vitro NK activity. Animals treated with anti-asialo GM1 antiserum were found to have a substantial decrease in the in vivo rate of clearance of tumor cells from the lungs. Furthermore, the adoptive transfer of a highly enriched population of LGL into NK-depressed animals 2 hr before tumor challenge, partially restored their cytotoxic activity against established cell lines in vitro and their ability to eliminate radiolabeled cells from the lungs. These results provide direct support for the hypothesis that NK cells are involved in in vivo resistance to tumors, particularly in the elimination of potentially metastatic tumor cells from the circulation and capillary beds.

Suppression of activity of mouse natural killer (NK) cells by activated macrophages from mice treated with pyran copolymer.

Treatment of young mice with pyran copolymer caused a substantial decrease in natural killer (NK) cell activity at 7 days. The decrease in cytotoxicity was associated with the presence of splenic suppressor cells, capable of inhibiting in vitro the NK activity of spleen cells from normal mice. The suppressor cells appeared to be macrophages, being plastic-adherent, phagocytic and radioresistant, and lacking demonstrable Thy 1.2 antigen. Sonicates or culture supernatants of adherent spleen cells from pyran-treated mice were also able to inhibit NK activity, suggesting that suppressor cells act by release of soluble factors.

Suppression of microtubule dynamics by binding of cemadotin to tubulin: possible mechanism for its antitumor action.

Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. Its primary mode of action has been unclear but is believed to involve an action on microtubules. We have found that cemadotin binds to tubulin and strongly suppresses microtubule dynamics. Scatchard analysis of cemadotin binding to tubulin indicated that there are two affinity classes of cemadotin-binding sites with Kd values of 19.4 microM and 136 microM. Cemadotin did not inhibit the binding of vinblastine to tubulin, and, conversely, vinblastine did not inhibit the binding of cemadotin to tubulin. By quantitative video microscopy of individual microtubules, we found that cemadotin strongly suppressed dynamic instability of microtubules assembled to steady state using bovine brain tubulin devoid of microtubule-associated proteins. It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.

Suppression of natural killer (NK) activity by splenic adherent cells of low NK-reactive mice.

Adherent cells with the ability to inhibit NK activity were found in the spleens of young 8-week-old SJL/J mice. Such suppressor cells were also found in the spleens of other low-NK-reactive strains, but not in the spleen of age-matched high-NK-reactive strains. These suppressor cells had the characteristics of macrophages, since they were plastic- and nylon-adherent, phagocytosed latex and iron particles, and were resistant to treatment with anti-Thy-1.2 serum plus complement. Their inhibitory activity appeared to be due to their production and/or release of soluble suppressive factor(s). Their suppressor activity was similar to that of pyran copolymer-activated macrophages, which were previously shown to be suppressive for cytotoxicity by NK cells. We suggest here that macrophages may be involved in the physiological regulation of NK activity and may contribute to the low NK activity in some strains of mice.

Synthesis of antitumor dendritic imides.

Dendritic imides were synthesized and evaluated as antitumor compounds. Compounds 8 and 11 showing a promising profile as inhibitors of lck but their antiproliferative activity against HT-29 was not so relevant.

Direct evidence for the role of LGL in the inhibition of experimental tumor metastases.

The role of NK cells in the control of the metastatic spread of tumor cells was studied. Rats pretreated with rabbit anti-asialo GM1 (anti-asGM1) serum exhibited a diminished ability to destroy circulating MADB106 mammary adenocarcinoma cells, which in turn caused an increased incidence of experimental pulmonary metastasis. The anti-asGM1 treatment caused a selective inhibition of NK activity without detectable effect on T cell-mediated immunity, and overall had no effect on the cytotoxic activity or numbers of alveolar macrophages (alv.M phi) or monocytes. The suggestion of a role for NK cells in resistance to metastases from the MADB106 tumor cells was confirmed by the adoptive transfer of 5 X 10(6) highly purified large granular lymphocytes (LGL) into NK-depressed animals 2 hr before tumor challenge. This transfer of LGL, highly enriched in NK activity, partially or fully restored the ability of these rats to inhibit the development of pulmonary metastases. This ability to adoptively transfer resistance to metastases appeared to be confined to the LGL population, because transfer of the same number of mature peripheral blood T cells had no effect on tumor development. These results provide the first unequivocal evidence that LGL, with high NK activity, are involved in in vivo resistance to tumors, particularly in the elimination of potentially metastatic tumor cells from the circulation and capillary beds.

Natural killer activity in the rat. IV. Distribution of large granular lymphocytes (LGL) following intravenous and intraperitoneal transfer.

Highly enriched populations of rat large granular lymphocytes (LGL) and T lymphocytes were prepared on discontinuous density gradients of Percoll, labeled with either 111In-oxine or 51Cr and injected either intravenously (iv) or intraperitoneally (ip) into normal syngeneic recipients. Following iv inoculation of labeled LGL or T cells into normal recipients, a large proportion of radioactivity (18 to 33%) was recovered within minutes in the lungs. By 2 to 4 hr following transfer, significantly more LGL (13.5%) than T cells (6.4%) remained in the lungs. This difference persisted through 48 hr (5.4 vs 0.8%). Decreasing levels of radioactivity in the lungs were accompanied by corresponding increases in counts in the spleen and liver. At early time points, a significantly higher proportion of T cells was found to distribute to the spleen, while labeled LGL persisted for longer periods in the blood as well as in the lungs. Following ip inoculation into normal recipients, there was a slow clearance of radiolabeled LGL and T cells from the peritoneal cavity, with less than 20% of the radiolabel found in peripheral organs by 24 hr. These results demonstrate a distribution pattern for LGL and T cells that resembles the previously reported proportions of these cells in various organs. In addition, these studies provide a firm basis for the formulation of further experiments to examine the usefulness of adoptive immunotherapy with LGL or immune T cells.