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P Q Patek

Publications and source records attributed to P Q Patek.

At least 19 recordsLinked to original sources
PubMed1995

The involvement of protein tyrosine kinase activity in a tumor necrosis factor resistance mechanism.

Certain cytokines activate pathways involving protein phosphorylation. Serine and threonine phosphorylation are most common, whereas tyrosine phosphorylation is a rare post-translational event, accounting for a very small percentage of phosphorylated amino acids. Nonetheless, protein tyrosine kinase activity is associated with several cell surface receptors and is involved in intracellular signaling. Here, we show that tumor necrosis factor (TNF) treatment of cells resistant to TNF-mediated lysis resulted in an increase in protein tyrosine kinase activity. Moreover certain TNF-resistant cell lines became sensitive to TNF-mediated cytolysis when treated with the inhibitors of protein tyrosine kinases, genistein, and herbimycin A. In contrast, genistein had no effect on the lysis a TNF-sensitive cell line. The increase in TNF-mediated lysis affected by genistein occurred only when it was present during TNF treatment, and the effect was maximal when the inhibitor was added 30 min after the TNF. These findings suggest that, in TNF-resistant cells, TNF activates a protein tyrosine kinase that contributes to the cell's resistance to lysis and this resistance mechanism does not function in the TNF-sensitive cell line.

Animals↗
PubMed1994

Transformed B cell lines express cell-associated tumor necrosis factor and mediate natural cytotoxic activity.

Natural cytotoxic (NC) activity is mediated by a cell-associated form of TNF-alpha. Here, we have characterized the NC-like properties of cloned murine transformed B cell lines. Several B cell lines cause the lysis of NC/TNF-alpha-susceptible target cells, but not NC/TNF-apha-resistant target cells. Furthermore, lysis of targets by B cell lines involves a nonsoluble (i.e., cell-associated) mechanism that is blocked by anti-TNF antibody. Target cells selected for resistance to a B lymphoma cell line expressing NC-like activity are resistant to splenic NC-mediated lysis, resistant to TNF-alpha-mediated lysis, and have increased tumorigenicity in BALB/c mice. Thus, these B cells lines may serve as cloned effectors which lyse cells using a cell-associated TNF-dependent mechanism analogous to NC lysis. Furthermore, it is possible that the expression of TNF by transformed B cells is autostimulatory and is involved in their malignant transformation.

Animals↗
PubMed1994

Inhibition of tumor necrosis factor-mediated lysis by spleen cell-conditioned medium.

A spleen cell-elaborated factor which inhibits both TNF and natural cytotoxic cell lytic activity has been described. Production of this factor is dependent on prostaglandins, and inhibitory activity is abrogated by protein synthesis inhibitors. Furthermore, it was demonstrated that the factor renders TNF sensitive cells refractory to TNF rather than acting on TNF directly. Here we show that the factor is produced by spleen cells from both BALB/c and C57bl/6 mice and inhibits TNF mediated lysis of various TNF sensitive cells. The inhibitory factor has a molecular weight greater than 30 kDal and is heat labile, suggesting it is a protein. Furthermore, the factor is produced by high density spleen cells (e.g. cells of the myeloid lineage). Although it is not clear which cell type(s) is responsible for the production of this factor, these findings suggest that high density spleen cells, which do not produce TNF, can modulate TNF lytic activity via this factor.

Animals↗
PubMed1991

In vitro selection of a cell line for resistance to lysis by tumor necrosis factor-alpha selects for reduced tumorigenicity.

Experimentally, TNF-alpha can mediate the hemorrhagic necrosis of certain tumors. Furthermore, evidence indicates that natural cytotoxic (NC) activity, a cell-mediated cytolytic activity that utilizes TNF-alpha in the lysis of target cells, is involved in preventing the outgrowth of certain NC/TNF-alpha-sensitive tumor cells. These observations raise the issue of whether soluble TNF-alpha normally serves as a tumor surveillance mechanism preventing the outgrowth of some tumors. To address this issue, we have used TNF-alpha to select TNF-alpha-resistant variants from the NC/TNF-alpha-sensitive mouse fibroblast cell line 10ME. Previously, we have demonstrated that 10ME is tumorigenic in immune-deficient mice but fails to form tumors in normal mice. Moreover, selection of NC-resistant variants from 10ME selects for both TNF-alpha resistance and tumorigenicity in normal mice. As cells that have been selected for NC resistance form tumors in normal mice, whereas the NC-sensitive parental cell line does not, it seems that escape from NC activity is sufficient to significantly increase the tumorigenic potential of the cell line. We show that the selection with TNF-alpha, although associated with NC resistance, does not increase the tumorigenic potential of 10ME cells but reduces it. Thus, NC activity appears to function as a mechanism to prevent tumor formation, and escape from NC activity allows for tumor formation; TNF-alpha does not have similar activity. Moreover, this suggests that NC activity is not equivalent to soluble TNF-alpha activity, but utilizes TNF-alpha more efficiently than soluble TNF-alpha, or NC activity involves both TNF-alpha and other effector mechanisms.

Animals↗
PubMed1989

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

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.

Animals↗
PubMed1989

Natural cytotoxic activity is not necessarily mediated by the release of tumour necrosis factor.

Natural cell-mediated cytotoxicity is mediated by a family of effector cells that express cytolytic activities distinct from those generally attributed to B cells, T cells and macrophages; it includes both natural killer (NK) and natural cytotoxic (NC) activities. There is now convincing evidence to show that NC activity, but not NK activity, is mediated by tumour necrosis factor (TNF). Further, it has been argued that it is the release of TNF, as a freely diffusible factor, that causes NC-mediated target lysis. Here, we present evidence that the admixture of NC-sensitive target cells and spleen cells, under conditions that result in NC-mediated target cell lysis, does not necessarily result in the release of freely diffusible TNF into the culture medium. Also, it is demonstrated that the procedures used do not result in inactivation or loss of significant amounts of TNF during the assay period, which might account for our failure to detect free TNF. These results suggest that NC activity is mediated by either a membrane-associated TNF activity, similar to that described for some of the lytic activity of activated macrophages, or by the release of TNF that is capable of acting only over a very short distance.

Animals↗
PubMed1988

Effect of mycoplasmas on natural cytotoxic activity and release of tumor necrosis factor alpha by spleen cells.

It has been reported that mycoplasma-infected cells are more sensitive to lysis by natural cytotoxic (NC) effector cells and that splenic NC cells release tumor necrosis factor (TNF-alpha) when they lyse sensitive target cells. Here we showed that spleen cells released TNF-alpha when they were incubated with NC-sensitive cells that were infected with mycoplasmas or when they were incubated with mycoplasmas alone, but did not release TNF-alpha when incubated with NC-sensitive cells that were not infected with mycoplasmas. Thus, in the presence of mycoplasmas, spleen cell cultures contain both NC effector cells and free TNF-alpha. Because NC-sensitive cells are also sensitive to free TNF-alpha, when mycoplasma-infected cells were incubated with spleen cells, they were lysed by the combination of NC cells and free TNF-alpha. When NC-sensitive cells that were not infected with mycoplasmas were incubated with spleen cells, they were lysed only by NC effector cells and thus appeared to be less sensitive than mycoplasma-infected cells. These results also suggested that the release of TNF-alpha may be part of a host protective response to mycoplasmas.

Animals↗
PubMed1988

Inhibition of tumour necrosis factor and natural cytotoxic cell lytic activities by a spleen cell-elaborated factor.

Natural cytotoxic (NC) cell lytic activity is mediated by tumour necrosis factor (TNF), a protein with potent cytolytic activity on certain target cells. TNF also appears to mediate a wide range of other important biological activities (e.g. interferon-like anti-viral activity, induction of granulocyte-monocyte colony-stimulating factor, mediation of endotoxin-induced shock). Evidence is presented here that spleen cells from normal, untreated mice produce a factor(s) that inhibits both NC and TNF cytolytic activity. The factor(s) has a molecular weight greater than 10,000. Since indomethacin inhibits production by spleen cells of the NC/TNF inhibitory factor, it is suggested that prostaglandins are involved in the regulation of its production. Additionally, these studies indicate that the factor(s) does not function by inactivation of either NC effectors or TNF molecules, or by inhibition of the binding of NC cells or TNF to targets. Instead, the data suggest that the factor(s) acts on the targets rendering them refractory to TNF binding. Moreover, since the factor(s) acts slowly and requires protein synthesis in the target to function, it appears that the inhibitory activity is mediated via de novo-synthesized proteins from the target cells. At present, it is not known whether such a factor functions in vivo, although it is conceivable that its in vivo role is to modulate the pathological potential of TNF by protecting certain cells from NC or TNF lysis.

Animals↗
PubMed1987

Humans express natural cytotoxic (NC) cell activity that is similar to murine NC cell activity.

The expression of natural cytotoxic (NC) activity is well defined in mice, but poorly defined in humans. In this paper we report that humans express naturally occurring cytotoxic cell activity that recognizes and lyses murine targets that are sensitive to lysis mediated by murine NC cells, but not murine targets that are resistant to lysis by murine NC cells. We present data showing that these naturally occurring human cytotoxic cells and murine NC cells have similar lytic mechanisms. Both the human cytotoxic cells described here, and murine NC cells, use tumor necrosis factor (TNF) to mediate the lysis of sensitive targets. Moreover, targets that resist murine NC-mediated lysis by a protein synthesis-dependent post-recognitive mechanism use a similar mechanism to prevent lysis mediated by naturally occurring human cytotoxic cells. In addition to the similarity of naturally occurring human cytotoxic cells and murine NC cells in their specificity and lytic mechanism, naturally occurring human cytotoxic cells and murine NC cells are also similar in that their activity is both associated with a monocyte lineage and age independent. Taken together, these data indicate that humans express NC activity.

Adult↗
PubMed1987

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms.

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

In vivo or in vitro selection for resistance to natural cytotoxic cell lysis selects for variants with increased tumorigenicity.

Experiments were designed to test the hypothesis that transformed cells that are NC sensitive must escape NC activity if they are to grow as tumors in normal individuals. NC-resistant variants were selected either in vivo or in vitro from NC-sensitive cell lines that grow as tumors in immunodeficient mice but not in syngeneic normal mice. The tumorigenicity of cloned NC-resistant variants was compared with the parental cell lines and to cell lines that went through the selection procedure, but after cloning remained NC sensitive. Cloned NC-resistant cell lines derived from tumors that developed in x-irradiated nude mice after the injection of an NC-sensitive cell line are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines derived from the same tumors are unable to grow as tumors in normal mice. Similarly, six of seven NC-resistant cloned cell lines independently isolated after in vitro selection for NC-resistance are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines isolated from the same in vitro selected populations are not tumorigenic in normal mice. Thus, either the in vivo or in vitro selection of NC-resistant cells selects for cells tumorigenic in normal mice; these findings, along with our previous observations that selection for cells tumorigenic in normal mice selects for NC resistance, provide compelling evidence that escape from NC activity is required before some transformed cells can grow as tumors in normal mice.

Animals↗
PubMed1985

Analysis of immune surveillance of sequentially derived cell lines that differ in their tumorigenic potential.

The immune surveillance hypothesis suggests that cancer evolves as a multistage process. Further, it predicts that cells intermediate on the pathway to cancer are susceptible to host protective mechanisms, and only those variants that are able to escape the protective mechanisms are able to grow as tumors. We have isolated, as lineages, fibroblast lines that express phenotypes predicted by the surveillance hypothesis. The lineages were derived by treating nontransformed cells (N-cells) with chemical carcinogens and by isolating transformed variants in vitro. From the transformants that are tumorigenic in immune-depressed ATXFL mice but rejected by normal mice (I-cells), variants were selected in vivo that had escaped the rejection mechanism(s) and had grown as tumors in normal mice (C-cells). Thus lineages were established comprised of sequentially derived cell lines with the following phenotypes: nontransformed, transformed but susceptible to host protective mechanisms, transformed and resistant to host protective mechanisms (i.e., N----I----C). With the use of in vivo cross-protection experiments, two independently derived I-cell lines were shown to express non-cross-reactive antigens that are not expressed by the parental nontransformed N-cells (i.e., transformation-associated antigens). The transformation-associated antigens are expressed at an equivalent level on the cells that are susceptible to rejection (i.e., I-lines) and those that have escaped rejection (i.e., C-lines). In addition, although the transformation-associated antigens expressed by I-cells induce an effective immune response capable of rejecting both the I-line and C-line, the expression of these antigens on C-cells does not induce an effective immune response. The role of host defense mechanisms in the rejection of these chemically transformed I-cells and the possible mechanisms by which C-cells escape rejection are discussed.

Animals↗
PubMed1983

Evidence that cytotoxic T cells and natural cytotoxic cells use different lytic mechanisms to lyse the same targets.

There is evidence that natural cytotoxic (NC) cells are in the T cell lineage. To lyse targets, cytotoxic T cells (TK) must recognize any of the myriad antigens plus syngeneic major histocompatibility complex (MHC) determinants. In spite of the evidence which indicates TK and NC are in the same lineage, NC cells can recognize few determinants (perhaps only one) and do not require recognition of MHC determinants. In addition to differences in the requirement for target recognition, in this report we show that TK cells and NC cells also use different lytic mechanisms to lyse the same targets. NC effectors initiate a lytic mechanism in NC-sensitive and NC-resistant targets. This lytic mechanism requires approximately 4 h before target lysis is apparent in NC-sensitive targets; it is inactivated by a protein synthesis-dependent counterlytic mechanism in NC-resistant targets. In contrast the TK lytic mechanism causes a rapid release of 51Cr from both NC-sensitive and NC-resistant targets and is not inhibited by the NC counterlytic mechanism present in NC-resistant cells. These findings lead to the conclusion that the mechanism used by NC cells to lyse targets is fundamentally different from that used by TK cells.

Animals↗
PubMed1983

An analysis of the sensitivity of somatic cell hybrids to natural killer cell- and natural cytotoxic cell-mediated lysis.

The analysis of the NK and NC sensitivity of somatic cell hybrids formed between parental cell lines that differ in their NK and NC sensitivity has shown the following. 1) The dominant expression of both NK and NC recognition determinants on target cells; 2) the dominant expression of two post-recognitive NC resistance mechanisms, one requiring protein synthesis and one being protein synthesis independent; and 3) the dominant expression of a post-recognitive NK resistance mechanism, which is protein synthesis independent. The post-recognitive protein synthesis-independent NC resistance mechanism confers no NK resistance and the post-recognitive NK resistance mechanism confers no NC resistance. Whether the post-recognitive protein synthesis-dependent NC resistance mechanism confers NK resistance remains open to question. The analysis of the hybrids indicates that transformed cells become sensitive to either NK- or NC-mediated lysis by losing their resistance to the lytic activity of these effector cells, and it appears that differentiation plays a role in determining whether NK or NC resistance will be lost upon transformation. A model is proposed in which the differentiation into a fibroblast associates the loss of NC resistance with transformation, whereas the differentiation into a lymphocyte associates the loss of NK resistance with transformation. Because the loss of NK resistance is not associated with the transformation of fibroblasts, they remain NK resistant, and because the transformation of lymphocytes is not associated with the loss of NC resistance, they remain NC resistant. This provides the basis for the target specificity exhibited by NK and NC effectors.

Animals↗
PubMed1982

In vivo surveillance of tumorigenic cells transformed in vitro.

Any theory of surveillance against cancer requires that cells susceptible to host protective mechanisms exist as intermediates on the pathway from normal to cancer. The failure to demonstrate a significant frequency of such intermediates as a result of chemical carcinogenesis has cast serious doubt on the validity of the surveillance hypothesis. Here we report the conditions in which such intermediates can be identified as the major class of transformed cells resulting from in vitro chemical carcinogenesis of a cloned fibroblastic cell line.

Benzopyrenes↗