Paul Q. Patek

Activity and dexamethasone sensitivity of natural cytotoxic cell subpopulations.

Abstract Natural cytotoxic (NC) activity is the natural cell-mediated cytotoxic activity of normal spleen cells directed at fibroblast targets. Treatment of spleen cells with dexamethasone, a synthetic glucocorticosteroid, reveals two populations of NC effectors: one population whose cytolytic activity is inhibited by dexamethasone (DEX S ) and another which is resistant to inhibition by dexamethasone (DEX R ). The short-term culturing of spleen cells results in a shift in the population of splenic NC effectors from predominantly DEX S to predominantly DEX R . This shift also occurs when spleen cells are treated with conditioned medium from which the short-term cultured DEX R populations were derived.

Cell lines cultured at high density are resistant to lysis by tumor necrosis factor and natural cytotoxic cells.

Abstract It has been suggested that natural cytotoxic (NC) cell activity and tumor necrosis factor (TNF), the molecular mediator of NC activity, are capable of protecting individuals against the progression of incipient tumors or could be useful in cancer therapy regimens. Much of this speculation arises as a result of in vitro studies, on a variety of tumor cells, demonstrating the cytolytic and cytostatic properties of NC and TNF activities. Here, evidence is presented showing that certain mouse fibroblast cell lines, generally considered sensitive to NC and TNF lysis, are quite resistant to these lytic activities when cultured at high cell density. Although a soluble factor that renders these same target cells resistant to NC and TNF lysis has been described, no such factor is involved in this high density-induced resistance. Rather, it appears that cell to cell contact of the targets is critical. Moreover, the induced resistance to NC and TNF lysis does not result from loss of either NC recognition determinants or TNF receptors by the target cells, but is the consequence of increased expression of a protein synthesis-dependent resistance mechanism. These observations raise the issue of the in vivo phenotype of cells characterized in vitro as sensitive to NC and TNF lysis. It is entirely possible that certain cells which are considered sensitive to NC and TNF activities are, in fact, resistant to these cytolytic activities when growing as tumors (i.e., at high cell density). Should this be the so, NC and TNF cytolytic activities may not function in vivo or may function only via some indirect means.

Cancer: A Problem in Somatic Cell Evolution

Our goal is to define steps required for a cell to grow as a cancer. The complexity of the factors involved in the development of cancer make it clear that the analysis of clinical malignancy will not reveal these steps. Since these steps cannot be analyzed at the single cell level in vivo we extrapolate from the experimental to the autochthonous situation using a theory that considers the development of cancer to be a problem in somatic cell evolution.

Dissociation of contact-noninhibition in vitro and tumorigenicity in vivo

For cell lines, the correlation of the in vitro expression of contact-inhibition to the in vivo expression of the nontumorigenic phenotype, and the correlation of the expression of contact-noninhibition (i.e., focus formation) to the expression of the tumorigenic phenotype are commonly used as a means to identify, in vitro, cells which presumably have undergone malignant transformation such that, if tested, they would grow as tumors in vivo. In this report we show that while this correlation is true for contact-noninhibited transformants induced by benzo(a)pyrene (BP), a DNA mutating and demethylating agent, it is not true for contact-noninhibited transformants induced by 5-azacytidine (AZC), a DNA demethylating agent which does not have mutagenic activity. The in vitro treatment of a contact-inhibited cell line with 5-azacytidine (AZC) results in the expression of contact-noninhibited transformants, which, in vitro quantitatively and qualitatively similar to those induced by BP but unlike BP, treatment with AZC does not result in the in vivo expression of tumorigenicity.

The cloned cell line L10A2.J expresses natural cytotoxic activity

The analysis of natural cytotoxicity (NC) has been hampered by the lack of cloned NC effectors. In studies reported here we show that the cloned cell line L10A2.J expresses properties similar to those of splenic NC effectors. L10A2.J cells lyse NC-sensitive targets, but do not lyse NC-resistant targets which are sensitive to lysis by natural killer (NK) or cytotoxic T lymphocytes. The mechanism by which L10A2.J cells lyse NC-sensitive targets is similar to the lytic mechanism of splenic NC effectors in that both result in the release of 51Cr from targets with a lag of 5-7 hr after effectors and targets are mixed. In addition, inhibition of protein synthesis during the in vitro assays of NC or L10A2.J lytic activity causes some NC-resistant targets to become sensitive to lysis by both NC and L10A2.J effectors. The only functional difference detected between L10A2.J and splenic NC effectors is in their recognition of targets. While L10A2.J and splenic NC effectors recognize many of the same targets (NC resistant and NC sensitive), L10A2.J, unlike splenic NC effectors, does not recognize the NK-sensitive cell line YAC-1.

DIFFERENT EFFECTORS AND EFFECTOR MECHANISMS ARE INVOLVED IN THE NATURAL CELL-MEDIATED LYSIS OF LYMPHOID AND FIBROBLAST TARGETS*

ABSTRACT We have found that NK-sensitive lymphoid cells as well as NC-sensitive and NC-resistant fibroblasts (normal and transformed) share NC-recognition determinants. Lymphoid target cell sensitivity or resistance to NK-lysis is determined by the presence or absence of NK-recognition determinants, respectively, while sensitivity or resistance of fibroblast targets to NC-lysis is determined by the level of a protein synthesis dependent counterlytic mechanism operating in the target cells. This counterlytic mechanism is absent from lymphoid targets.