Characterization of human non-small cell lung cancer (NSCLC) cell lines for expression of MHC, co-stimulatory molecules and tumor-associated antigens.

A panel of 31 long-term non-small cell lung cancer (NSCLC) cell lines was examined for the expression of protein and/or mRNA transcripts for 11 distinct immune response related molecules or tumor associated antigens (TAA). To assess whether cytokine stimulation might up-regulate expression of the genes of interest, cells were cultured in 500 U/ml of gamma-interferon (gamma-IFN) for 48-72 h prior to analysis. Major histocompatibility complex (MHC) Class I antigens were detected by indirect immunofluorescence and were constitutively expressed on all of the cell lines. The average of the mean fluorescence intensity (MFI) measured 222+/-22. gamma-IFN stimulation produced a significant increase to 482+/-36. For MHC Class II only 7/31 cell lines (23%) exhibited constitutive expression, while gamma-IFN treatment had a dramatic effect and yielded 18/31 (58%) positive cell lines. The co-stimulatory molecules CD80 and CD86 were examined by direct immunofluorescence for cell surface expression and RT-PCR amplification for mRNA. CD80 protein was not detected at all, while an insignificant percentage of cells were positive (mean 2%) for CD86 in all cell lines tested. gamma-IFN had no apparent effect on CD80 or CD86 protein expression. Constitutive CD80 or CD86 mRNA levels were observed in 45 and 61% of the NSCLC lines, respectively. These percentages increased to 77% and 90% with gamma-IFN. Cell surface phenotypic analysis for TAA revealed positive populations in 28/31 cell lines (90%) for Her-2/neu, 18/31 (58%) for CEA and 8/31 (26%) for GD-2, with gamma-IFN having no effect. After gamma-IFN stimulation, RT-PCR amplification for Mage-1, -2, -3 and WT-1 detected mRNA in 33%, 33%, 44% and 70% of the cell lines, respectively. Overall, gamma-IFN stimulation led to the up-regulation of MHC Class I molecules and class II molecules as well as CD80 and CD86 mRNA transcripts. This survey represents the first comprehensive analysis of NSCLC cell lines for a variety of molecules that could play an important role in the generation of an NSCLC anti-tumor CD8+ cytotoxic T lymphocyte (CTL) response.

Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease.

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.

High-dose naltrexone and liver function safety.

Studies have found naltrexone useful in the treatment of diseases other than opiate addiction in which endogenous opioids presumably play a role, such as alcoholism and eating disorders. Some of these studies involve high doses (100-200 mg bid). Because investigational studies with high doses (300 mg/day) reported clinically significant increases in liver enzyme levels, the authors measured a spectrum of liver function parameters in response to high doses of naltrexone in a double-blind, crossover trial (100 mg bid) followed by an open-label period (200 mg bid). They observed no adverse clinical or laboratory changes in liver function in association with high-dose naltrexone therapy in eating disorders.

Selective elimination (purging) of contaminating malignant cells from hematopoietic stem cell autografts using recombinant adenovirus.

In this work we have explored the use of adenoviral vectors for the purging of cancer cells from hematopoietic stem cell (HSC) autografts. We showed that a recombinant adenovirus expressing the herpes simplex-1 thymidine kinase gene (AD-tk) plus ganciclovir (GCV) killed HELA cells more effectively than did GCV alone. HELA cells were then mixed with human HSCs and exposed to AD-tk/GCV. AD-tk/GCV reduced the number of HELA colonies to 4% of control values, with no detectable reduction in the hematopoietic progenitor, colony forming unit-granulocyte/monocyte (CFU-GM). Similar studies of the JB6 non-Hodgkins lymphoma cell line showed a reduction to 5% of controls; studies of MCF-7, a breast carcinoma cell line, showed a reduction to 30% of controls, with no CFU-GM toxicity. Thus, AD-tk mediated selective killing of contaminating tumor cells. We also evaluated a recombinant adenovirus encoding the tumor suppressor gene p53 (AD-p53). AD-p53 was able to selectively kill all three cell lines (reducing tumor colonies approximately 100-fold) without any toxicity to CFU-GM. Although both AD-tk/GCV and AD-p53 were effective in these experiments, AD-p53 seemed to be more potent. Adenoviral vectors show promise for selectively targeting cancer cells that contaminate HSC autografts.

Differential effects of oral losartan and enalapril on local venous and systemic pressor responses to angiotensin I and II in healthy men.

This double-blind, placebo-controlled crossover study was designed to differentiate the pharmacodynamic effects of the angiotensin II receptor antagonist losartan from the angiotensin converting enzyme inhibitor enalapril. Effects of placebo, enalapril (10 mg), and losartan (20 and 100 mg) on local venous and systemic pressor responses to angiotensin I and II were compared in eight healthy male subjects. Treatments were administered orally approximately 4 hours before agonist infusions into a dorsal hand vein. Local changes in hand vein diameter and systemic blood pressure were monitored during the infusions. The 100 mg dose of losartan attenuated local venoconstrictor and systemic pressor responses to angiotensin I and II. In contrast, enalapril blocked only responses to angiotensin I. Both losartan and enalapril increased plasma renin concentration compared with placebo. These results are consistent with direct antagonism of angiotensin II receptors by losartan and with indirect effects of enalapril through inhibition of angiotensin converting enzyme.

Genetic control of the target structures recognized in hybrid resistance.

Hybrid resistance of lethally irradiated (C57BL/6 X DBA/2)F1 and (C57BL/10 X C3H)F1 hybrid mice to the engraftment of parental C57BL/6 or C57BL/10 bone marrow cells is controlled by the H-2-linked Hh-1 locus. This resistance can be specifically blocked or inhibited by the injection of irradiated spleen cells from lethally irradiated, marrow reconstituted donor mice of certain strains. By testing the ability of regenerating spleen cells from various donor strains to block the resistance, we studied the genetic requirements for the expression of putative cell-surface structures recognized in hybrid resistance to H-2b marrow cells. Strains of mice bearing informative intra-H-2 or H-2/Qa-Tla recombinant haplotypes provided evidence that the Hh-1 locus is located telomeric to the H-2S region complement loci and centromeric to the H-2D region class I locus in the H-2b chromosome. Two mutations that affect the class I H-2Db gene have no effect on Hh-1b gene expression. The H-2D region of the H-2s haplotype contains an allele of the Hh-1 locus indistinguishable from that of the H-2Db region, as judged by the phenotypes of relevant strains and F1 hybrids. Collectively these data indicate that the Hh-1 locus is distinct from the class I H-2D (L) locus in the H-2b or H-2s genome, and favor the view that the expression or recognition of the relevant determinants is not associated with class I gene products.