Wilbert Matthews

Characteristics of nine newly derived mesothelioma cell lines

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

Serum ribonucleases in cancer: Relation to tumor histology

Serum alkaline ribonuclease (SRNase) levels were determined in 49 normal adults and 94 patients with clinically operable malignancies. The mean SRNase levels were higher in 22 patients with adenocarcinomas (P < .003) and 28 patients with squamous carcinomas (P < .01) than in controls. These levels were also similar in patients with primary tumors only and those with metastases. The RNase levels of 21 patients with sarcomas and 23 patients with melanomas, however, did not differ from controls. The increased SRNase levels that occur early in the clinical evolution of adenocarcinomas and squamous carcinomas indicate that this change may have biological significance in the development of these malignancies. The correlation of increased SRNase levels with tumor histology may explain previous conflicting reports concerning abnormal levels of the enzyme in cancer patients.

Spin trap protection from tumor necrosis factor cytotoxicity

Tumor necrosis factor (TNF) facilitates superoxide production, and spin traps may detoxify superoxide by acting as superoxide dismutase mimics. We investigated the ability of a stable nitroxide spin trap, TEMPOL, to protect TNF-sensitive cells from exogenously added TNF. WEHI or L929 cells were incubated with TNF (500 units/ml) for 18 hr either simultaneously with 0 to 8 mM TEMPOL or with the TEMPOL added at varying intervals after TNF exposure. A dose-dependent increase in survival was noted in the TEMPOL-treated cells, with 92 +/- 2% survival of WEHIs treated with 4 mM TEMPOL compared to 26 +/- 1% survival for non-TEMPOL-exposed cells (P2 less than 0.01). Significant increases in survival could be accomplished with as late as 15-hr delayed addition of the compound. The mechanism of protection does not seem to involve newly synthesized protein, and Northern blot analysis revealed that TEMPOL does not induce the genes for MnSOD or Cu-ZnSOD. The ability of TEMPOL to protect against TNF injury, even when exposure is delayed, may prove useful in conditions thought to be associated with free radical-lymphokine interactions such as ischemia-reperfusion, oxygen toxicity, or sepsis.

In vitro photodynamic therapy of human lung cancer

Photodynamic therapy (PDT) using a dihematoporphyrin ether (DHE) sensitizes malignant cells to damage by 630-nm light. This study investigated in vitro PDT sensitivity of human lung cancer cells (A549) and those factors which influence cell survival as determined by the colony formation assay. After incubation for 2, 4, or 6 hr with [DHE] of 2.5, 25, or 50 micrograms/ml, A549 received red light at dose rates of 0.27 or 0.09 mW/cm2 and energies of 0-250 mJ/cm2. Neither 630-nm light alone nor DHE alone affected cell survival. A dose rate of 0.27 mW/cm2 required less energy than 0.09 mW/cm2 for 90% cytotoxicity (180 mJ/cm2 vs 250 mJ/cm2, P less than 0.05). The energy required for 90% cytotoxicity with 25 micrograms/ml [DHE] was dependent on DHE incubation time (2 hr, 90% cytotoxicity not reached; 4 hr, 116 mJ/cm2; 6 hr, 69 mJ/cm2; P less than 0.05). In contrast, cellular [DHE] as measured by fluorescence, plateaued after 2 hr of incubation. Fluorescence microscopy revealed a time-dependent redistribution of fluorescence from the cell membrane to perinuclear and intracytoplasmic organelles. A 99% cytotoxicity required significantly less energy as [DHE] was increased (2.5 micrograms/ml, no cytotoxicity; 25 micrograms/ml, 243 mJ/cm2; 50 micrograms/ml, 111 mJ/cm2; P less than 0.05). Intracellular [DHE] was directly dependent on the incubating media [DHE] (2.5 micrograms/ml, 0.09 +/- 0.01 micrograms/10(6) cells; 25 micrograms/ml, 0.80 +/- 0.07 micrograms/10(6) cells; 50 micrograms/ml, 1.31 +/- 0.11 micrograms/10(6) cells; P less than 0.05). PDT cytotoxicity was inversely proportional to concentration of serum in the DHE media. These data illustrate that lung cancer in vitro is sensitive to PDT and is influenced by dose rate, energy input, and DHE environmental manipulations. These factors may be important in increasing the efficiency of PDT of thoracic malignancies in vivo.

Alterations in macrophage free radical and tumor necrosis factor production by a potassium channel activator

The potassium channel activator nicorandil, under evaluation for antianginal management, has been shown to decrease neutrophil respiratory burst. Since our laboratory has demonstrated that reactive oxygen species (ROS) increase tumor necrosis factor (TNF) production, we hypothesized that nicorandil might decrease TNF production from a lipopolysaccharide (LPS) challenge via reduction of respiratory burst. Macrophage viability and TNF production were determined after an 18-hr exposure to 5.0 micrograms/ml LPS and varying concentrations of nicorandil. Nicorandil was not toxic to macrophages below 12 mM (94 +/- 3% viability versus control) and decreased ROS and TNF production. Intracellular superoxide production decreased from 164 +/- 24 OD550 to 99 +/- 6 OD550 with 10 mM nicorandil and extracellular superoxide decreased from 3108 +/- 111 to 1760 +/- 210 nM. Hydrogen peroxide production was also decreased by 10 mM nicorandil. TNF production in response to 5 micrograms/ml LPS decreased from 6.8 +/- 0.6 to 2.7 +/- 0.4 ng/ml with 10 mM nicorandil. Northern and slot blot analyses demonstrate that nicorandil acts at a post-transcriptional site. These data imply that nicorandil decreases macrophage TNF production from an LPS challenge, possibly through a reduction in respiratory burst. Such compounds may prove useful in the treatment of conditions thought to be associated with free radical-lymphokine interactions such as ischemia-reperfusion injury, oxygen toxicity, adult respiratory distress syndrome, and septic shock.

Serum ribonuclease in urologic cancer Relation to host immunocompetence

Abstract Increased circulating levels of S-RNase (serum ribonuclease), an enzyme with immunosuppressive properties, were demonstrated in 14 of 19 patients with prostatic carcinoma, 6 of 6 patients with renal cell carcinoma, 5 of 6 patients with bladder carcinoma, but only 1 of 8 patients with testis tumors. A significant correlation between S-RNase levels and the extent of tumor was demonstrated in patients with prostatic carcinoma, but not in patients with other urologic malignant conditions. Moreover, a significant correlation was demonstrated between S-RNase and host cell-mediated immunocompetence as measured by delayed cutaneous hypersensitivity to DNCB (dinitrochlorobenzene). The results suggest that the S-RNase assay may be of clinical use in experimental treatment programs to reflect fluctuations of tumor status when subtle effects on tumor growth may be obscured by the advanced tumor state.

Recruitment of inflammatory cells to a tumor deposit potentiates the immunotherapeutic effects of interleukin-2.

SummaryInterleukin-2 (IL-2) is a potent immunotherapeutic agent in murine models of intraperitoneal, pulmonary, and hepatic tumor implantation. Because of the systemic toxicity documented at doses of IL-2 required to control tumor growth, potentiation of the effects of low dose IL-2 is an important problem in immunotherapy. To address this problem, we attempted to recruit lymphocytes into a tumor mass. Allogeneic P185 (H-2d) tumor was mixed with MCA-105 (H-2b) tumor and injected s. c. into C57BL/6 (H-2b) mice. Mice were treated with 50,000 units of IL-2 twice daily from day 0 to day 6. When IL-2 alone was used to treat s. c. tumor, there was no reduction in the size of tumor implants. When allogeneic tumor was mixed with syngeneic tumor, there was a reduction in tumor size at the high dose of allogeneic tumor but not at the low dose. When allogeneic tumor was mixed with syngeneic tumor and the mice treated with IL-2, the immunotherapeutic effects of IL-2 were markedly increased. These studies show that an immune response to alloantigens, generated within tumor tissue can augment the immunotherapeutic effects of exogenous IL-2.

Early antigen elimination by cytotoxic t cells results in diminished cellular immune responses to allogeneic tumor.

Abstract Previous reports have suggested that repeated alloantigenic challenge increases humoral responses to alloantigens, but may cause decreasing cellular responses. We stimulated BALB/c (H-2 d ) mice with intraperitoneal EL-4 tumor (H-2 b ) and serially assessed cytotoxic responses in spleen and peritoneal lymphocytes using 51 Cr-labeled EL-4 target cells. We observed that cytotoxicity generated in the spleen of nonimmune BALB/c mice was much greater than that in immunized mice; similar peak responses were generated in peritoneal lymphocytes in normal and immunized hosts. Complement-mediated cytotoxicity was not necessary for diminished splenic responses in hyperimmune hosts, for the same phenomenon was seen in the Hzl anti-BL/6 system which is free of humoral responses. Irradiation (2000 rad) of the EL-4 tumor challenge prevented tumor cell proliferation and markedly reduced splenic responses in nonimmune mice. We suggest that cytotoxic cells suppressed further generation of cyto-toxicity; by effecting an early elimination of tumor inoculum, tumor proliferation was abrogated and dose of cellular antigen was, consequently, markedly reduced.

Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis

Abstract Irradiated BALB/c spleen-cell populations actively cytotoxic to BL/6 alloantigens (modulator cells) were capable of suppression of the in vitro generation of BALB/c anti-BL/6 cellular cytotoxicity. This suppression was abrogated by anti-θ serum plus complement. The suppression was dose dependent on the number of modulator cells and correlated directly with the magnitude of their cytotoxicity. By varying the number of stimulator cells, specific suppression for a relevant stimulator cell and nonspecific suppression for an irrelevant stimulator cell were demonstrated in the same cultures. These data suggest that cytotoxic cells caused specific suppression in mixed lymphocyte culture by lysing stimulator cells although evidence for other nonspecific suppressor factors was seen. A model was proposed suggesting that cell populations possessing high levels of cytotoxicity may feed back negatively on an ongoing immune response by competing with proliferating T cells for cellular antigen.

Alloantigen-activated lysis of syngenic tumor augmented by allogenic lymphocytes as cold targets.

In the past, most work in tumor immunology involved attempts to demonstrate tumor-specific transplantation antigens; however, recent in vitro work has shown that it is not necessary to sensitize to a specific tumor antigen to achieve lysis of syngenic tumor cells. Responder spleen cells from B6AF1 mice (H-2a,b) were sensitized in mixed lymphocyte cultures (MLC) to alloantigens on spleen cells from B10.D2 (H-2d) mice. In cell-mediated lympholysis (CML) assays the relevant allogenic P815 (H-2d) tumor targets and B10.D2 lymphoblast targets were lysed. In addition, the semisyngenic tumor target EL-4 (H-2b) was lysed but RDM-4 (H-2k) was not. B10.BR (H-2k), C57BL/6 (H-2b) and B6AF1 lymphoblasts were not lysed. If lymphocytes were added as cold targets to the CML assay, B10.D2 lymphocytes completely absorbed lytic activity when B10.D2 lymphoblasts were the target. If B10.D2 lymphocytes were added when P815 was the target, lysis was reduced but could not be abolished. When B10.D2 lymphocytes were added with EL-4 as a target, lysis doubled. These experiments show that the neoplastic determinant on tumor cells recognized by alloantigen-activated lymphocytes does not cross-react with stimulator cell alloantigen and is not an alien histocompatibility antigen. These observations should be considered when in vivo attempts are made to control tumor with in vitro activated lymphocytes; transfer of not only in vitro activated cells but also allogenic stimulator cells to the tumor site may be necessary for maximal tumor destruction.