PTTG1 Levels Are Predictive of Saracatinib Sensitivity in Ovarian Cancer Cell Lines.

Src kinase is recognized as a key target for molecular cancer therapy. However, methods to efficiently select patients responsive to Src inhibitors are lacking. We explored the sensitivity of ovarian cancer cell lines to the Src kinase inhibitor saracatinib to identify predictive markers of drug sensitivity using gene microarrays. Pituitary tumor transforming gene 1 (PTTG1) was selected as a potential biomarker as mRNA levels were correlated with saracatinib resistance, as well as higher PTTG1 protein expression. PTTG1 expression was correlated with proliferation, cell division, and mitosis in ovarian cancer tissues data sets. In sensitive cell lines, saracatinib treatment decreased PTTG1 and fibroblast growth factor 2 (FGF2) protein levels. Downregulating PTTG1 by siRNAs increased saracatinib sensitivity in two resistant cell lines. Our results indicate PTTG1 may be a valuable biomarker in ovarian cancer to predict sensitivity to saracatinib, and could form the basis of a targeted prospective saracatinib trial for ovarian cancer.

A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop.

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines.

BACKGROUND: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas.

HER-2/neu gene amplification and cell surface overexpression are important factors in breast cancer for prognosis and prediction of sensitivity to anti-HER-2/neu monoclonal antibody therapy. In lung cancer, the clinical significance of HER-2/neu expression is currently under evaluation. We investigated 238 non-small lung carcinomas for HER-2/neu protein overexpression by immunohistochemistry using the HercepTest. We found 2+ or 3+ overexpression in 39 patients (16%), including 35% in adenocarcinomas and 20% in large cell carcinomas, but only 1% of squamous cell carcinomas. Marked (3+) overexpression was uncommon (4%). The association between protein expression and gene copy number per cell, as determined by fluorescence in situ hybridisation assay, was investigated in 51 of these NSCLC tumours. Twenty-seven tumours (53%) were negative by both tests. Marked (3+) protein expression and gene amplification were present in only 4% of samples. In 11 tumours (21%), gene gain was accompanied by chromosomal aneusomy and did not result in high protein levels while in 7 (14%) the score 2+ was associated with maximum number of signals per cell <9. The prognostic implication of HER-2/neu protein expression was studied in 187 surgically resected tumours. No statistical difference in survival was observed comparing patients with positive (2+/3+) and negative tumours (0/1+), although 3+ patients showed a tendency to shorter survival. The therapeutic implications of protein expression and gene amplification in lung cancer need to be examined in prospective clinical trials.

Expression of Her-2/neu in human lung cancer cell lines by immunohistochemistry and fluorescence in situ hybridization and its relationship to in vitro cytotoxicity by trastuzumab and chemotherapeutic agents.

Overexpression of the Her-2/neu oncogene and receptor protein was reported in approximately 20% of breast cancers and was associated with a poor prognosis. Her-2/neu expression was a predictor for response to trastuzumab, a monoclonal antibody that recognizes the Her-2/neu cell surface receptor. Data regarding the expression of Her-2/neu in lung cancer are far more limited, and there is little information regarding the influence of Her-2/neu expression and response to trastuzumab alone or in combination with chemotherapeutic agents. In this report we evaluated Her-2/neu gene expression by fluorescence in situ hybridization (FISH) and the cell surface expression of the Her-2/neu receptor by immunohistochemistry using the HercepTest and by FACS analysis in 31 lung cancer cell lines with 5 breast cancer cell lines as controls. By FACS, we found Her-2/neu overexpression (mean fluorescence intensity >8) in 2 of the 22 non-small cell lung cancer (NSCLC) cell lines (9%), none of 11 small cell lung cancer (SCLC) cell lines, and 4 of 5 breast cancer cell lines. A positive HercepTest (2+ or 3+) was found in 6 of 19 NSCLC cell lines (26%, 2+; 5%, 3+), 1 of 3 SCLC cell lines (33%), and 4 of 5 breast cancer cell lines (80%). One of 6 NSCLC cell lines examined (17%) had gene amplification with >32 copies of Her-2/neu/cell and had homogeneous staining regions. One NSCLC cell line had a maximum of 14 copies of Her-2/neu/cell, and 3 had modest increases in Her-2/neu gene copy number without gene amplification (maximum 5-8 copies/cell). None of the SCLC cell lines had more than a maximum of 4 copies/cell, whereas the 2 breast cancer cell lines had maximum Her-2/neu copy numbers of 80 and 5, respectively. Aneusomy rather than true amplification was the major cause of increased Her-2/neu expression in most of the NSCLC cell lines. There was a strong correlation when the results of fluorescence-activated cell sorter, HercepTest results, and FISH were compared in pairs. Furthermore, Trastuzumab produced a G(1) cell cycle arrest and growth inhibition only in cell lines expressing Her-2/neu. The IC(50) for growth inhibition was correlated with cell surface Her-2/neu expression. The combination of trastuzumab and chemotherapeutic agents produced more than additive growth inhibition in cell lines expressing Her-2/neu, but the level of additivity was not related to the amount of Her-2/neu expression. These data indicate that trastuzumab alone and in combination with chemotherapeutic agents should be tested in NSCLC patients and that Her-2/neu should be assessed by both immunohistochemistry and FISH methods in these studies to determine which test is the best predictor of outcome.

Altered HOX and WNT7A expression in human lung cancer.

HOX genes encode transcription factors that control patterning and cell fates. Alterations in HOX expression have been clearly implicated in leukemia, but their role in most other malignant diseases remains unknown. By using degenerate reverse transcription-PCR and subsequent real-time quantitative assays, we examined HOX expression in lung cancer cell lines, direct tumor-control pairs, and bronchial epithelial cultures. As in leukemia, genes of the HOX9 paralogous group and HOXA10 were frequently overexpressed. For HOXB9, we confirmed that elevated RNA was associated with protein overexpression. In some cases, marked HOX overexpression was associated with elevated FGF10 and FGF17. During development, the WNT pathway affects cell fate, polarity, and proliferation, and WNT7a has been implicated in the maintenance of HOX expression. In contrast to normal lung and mortal short-term bronchial epithelial cultures, WNT7a was frequently reduced or absent in lung cancers. In immortalized bronchial epithelial cells, WNT7a was lost concomitantly with HOXA1, and a statistically significant correlation between the expression of both genes was observed in lung cancer cell lines. Furthermore, we identified a homozygous deletion of beta-catenin in the mesothelioma, NCI-H28, associated with reduced WNT7a and the lowest overall cell line expression of HOXA1, HOXA7, HOXA9, and HOXA10, whereas HOXB9 levels were unaffected. Of note, both WNT7a and beta-catenin are encoded on chromosome 3p, which undergoes frequent loss of heterozygosity in these tumors. Our results suggest that alterations in regulatory circuits involving HOX, WNT, and possibly fibroblast growth factor pathways occur frequently in lung cancer.

Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines.

Non-small cell lung cancer (NSCLC) cells have constitutively high expression of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) 2. These NSCLC cells also have increased prostaglandin expression (PGE2). Many lung cancers also express 12-lipoxygenase RNA and 12-lipoxygenase protein and biosynthesize 12(S)-hydroxyeicosatetraenoic acid, which correlates with their metastatic potential. Several studies have demonstrated that COX-1 and COX-2 inhibitors could inhibit the in vitro growth of human lung cancer cell lines. In this report, we evaluated the growth-inhibitory effects of sulindac sulfide, a COX-1 and COX-2 inhibitor; exisulind (sulindac sulfone), a novel proapoptotic agent that does not inhibit COX enzymes; and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor on human lung cancer cell lines. We compared these effects with those of 13-cis-retinoic acid, a chemoprevention agent, and with the cytotoxic chemotherapeutic agents paclitaxel and cisplatin, alone or in combination. Our goal was to develop new chemoprevention and treatment strategies. Each of the six agents tested inhibited the in vitro growth of three NSCLC and three SCLC cell lines at the highest concentration. Paclitaxel was the most potent agent (IC50 = 0.003-0.150 microM); sulindac sulfide, NDGA, and 13-cis-retinoic acid had intermediate potency (IC50 = 4-80 microM), and cisplatin and exisulind were the least potent (IC50 = 150-500 microM). Combination studies showed synergistic interactions for sulindac sulfide, exisulind, and NDGA with paclitaxel, cisplatin, and 13-cis-retinoic acid, regardless of drug-resistance phenotype. At high concentrations, the combination of 13-cis-retinoic acid and each of the five other drugs resulted in a strong synergistic effect. These studies provide a rationale for chemoprevention (exisulind +/- retinoic acid +/- NDGA) and therapeutic (exisulind +/- paclitaxel +/- cisplatin) studies in patients at risk for, or with, lung cancer.

Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo.

Many lung cancers are stimulated by an autocrine/paracrine system of neuroendocrine peptide hormones. Attempts to block this autocrine growth pathway by interactions with specific ligand-receptor binding using monoclonal antibodies and peptide-specific antagonists have been largely unsuccessful because of the heterogeneity of hormone production and receptor expression. In the normal lung, neutral endopeptidase (NEP; CD10, CALLA, enkephalinase, and EC 3.4.24.11) plays a physiological role in degrading biologically active peptides, including all peptides implicated in autocrine growth stimulation of lung cancer. Cigarette smoke decreases the activity of NEP, indicating that the lack of NEP contributes to the dysregulation of the peptide autocrine system. The cloning of the human NEP gene allowed for production of sufficient quantities of recombinant NEP (rNEP) to evaluate its role in inhibiting the growth of lung cancer cells. In this study, we evaluated the ability of rNEP to inactivate the peptides involved in lung cancer signal transduction and to inhibit the growth of lung cancer cells as well as normal lung cells in vitro and in vivo in athymic nude mice. We showed that the growth inhibition of lung cancer cells by rNEP was related to the dose and schedule. Continuous exposure to high doses was required for growth inhibition. These studies confirm the importance of NEP in this autocrine pathway.

Reversal of anticancer multidrug resistance by the ardeemins.

Two "reverse prenyl" hexahydropyrroloindole alkaloids, 5-N-acetylardeemin and 5-N-acetyl-8-demethylardeemin, were evaluated as reversal agents in cells exhibiting a multidrug resistant (MDR) phenotype. These ardeemins (i) reversed drug resistance to vinblastine (VBL) or to taxol as much as 700-fold at relatively noncytotoxic concentrations in vitro; (ii) as a single agent at high concentrations killed MDR cells more efficaciously than the respective parent wild-type cells; and (iii) exhibited strong synergistic effects with doxorubicin (DX) and VBL against the growth of MDR neoplastic cells, and to a lesser extent, of the parent wild-type cells. Mechanistic studies showed that photoaffinity labeling of P-glycoprotein (Pgp) with [3H] azidopine was competitively inhibited by the ardeemins. Resistance to DX in MDR-[Pgp+ and MDR-associated protein (MRP)+], MDR-Pgp+, lung resistance protein (LRP)+-expressing, and wild-type lung cancer cells were reversed 110- to 200-fold, 50- to 66-fold, 7- to 15-fold, and 0.9- to 3-fold, respectively, by 20 microM of the ardeemins. Moreover, these compounds increased the intracellular accumulation of VBL and markedly decreased its efflux. Finally, in vivo combination studies demonstrated that nontoxic doses of the ardeemins with DX significantly improved the chemotherapeutic effects in B6D2F1 mice bearing DX-resistant P388 leukemia, and nude mice bearing human MX-1 mammary carcinoma xenografts. The above features indicate that the ardeemins may have utility in the therapy of cancer.

Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogen-activated protein kinase signaling and inhibits small cell lung cancer growth.

Transformed growth of human small cell lung cancer (SCLC) is mediated by autocrine signaling through multiple G protein-coupled neuropeptide receptors. To define the role of Gq and its effector, phospholipase Cbeta (PLCbeta), in SCLC growth, we expressed a COOH-terminal fragment of PLCbeta1 (PLCbetaCT) that is catalytically inactive and is predicted to behave as a competitive inhibitor of Gq signaling. Using endogenous muscarinic receptors as indicators of Gq-coupled receptor signaling status, we observed that stable expression of PLCbetaCT in NCI-H345 SCLC cells significantly inhibited muscarinic receptor-mediated phospholipase C activation and intracellular Ca2+ mobilization. In addition, PLCbetaCT expression reduced the basal activity of protein kinase C as well as the receptor-stimulated activity of the extracellular signal-regulated kinases, consistent with the sequential requirement for Gq, PLCbeta, and protein kinase C in the regulation of the extracellular signal-regulated kinases by neuropeptide and muscarinic receptors in SCLC. By contrast, muscarinic agonist stimulation of the c-Jun NH2-terminal kinases was not inhibited in SCLC cells expressing PLCbetaCT, indicating that other G proteins such as the G12,13 family members mediate c-Jun NH2-terminal kinase activation by neuropeptides and muscarinic agonists. Finally, soft agar colony formation by the SCLC cells expressing PLCbetaCT, but not growth in suspension culture, was markedly reduced, indicating that signaling through Gq and PLCbeta by autocrine-signaling neuropeptide receptors is a dominant pathway involved in the transformed growth of SCLC.

Neutral endopeptidase: variable expression in human lung, inactivation in lung cancer, and modulation of peptide-induced calcium flux.

Neutral endopeptidase (NEP; CALLA, CD10, EC 3.4.24.11) is a cell surface endopeptidase that hydrolyses bioactive peptides, including the bombesin-like peptides, as well as other neuropeptides. Bombesin-like peptides and other neuropeptides are autocrine growth factors for both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Low expression of NEP has been reported in SCLC and NSCLC cell lines. NEP inhibition has been shown to increase proliferation in one cell line. To date, NEP expression has not been quantitatively evaluated in normal adult lung, SCLC or NSCLC tumors, paired uninvolved lung from the same patient, or in other pulmonary neoplasms such as mesotheliomas and carcinoids. We examined the expression of NEP in these tissues and human cell lines using immunohistochemistry, flow cytometry, enzyme activity, ELISA, Western blot, and reverse transcription (RT)-PCR. Uninvolved lung tissue from different individuals displayed considerable variation in NEP activity and protein. By immunohistochemistry, NEP expression was detectable in alveolar and airway epithelium, fibroblasts of normal lung, and in mesotheliomas, whereas it was undetectable in most SCLC, adenocarcinoma, squamous cell carcinoma, and carcinoid tumors of the lung. NEP activity and protein levels were lower in all SCLC and adenocarcinoma tumors when compared to adjacent uninvolved lung, often at levels consistent with expression derived from contaminating stroma. NEP expression and activity were reduced or undetectable in most SCLC and lung adenocarcinoma cell lines. NEP mRNA by RT-PCR was not expressed or was in low abundance in the majority of lung cancer cell lines. The majority of lung tumors did not express NEP by RT-PCR as compared with normal adjacent lung. In addition, recombinant NEP abolished, whereas an NEP inhibitor potentiated, the calcium flux generated by neuropeptides in some lung cancer cell lines, demonstrating potential physiological significance for low NEP expression. NEP, therefore, is a signal transduction and possibly a growth modulator for both SCLC and NSCLC, emphasizing the role of neuropeptides in the pathogenesis of the major histological forms of lung cancer.

Discordant signal transduction and growth inhibition of small cell lung carcinomas induced by expression of GTPase-deficient G alpha 16.

Small cell lung carcinoma (SCLC) accounts for 20-25% of primary lung cancers and is rapidly growing, widely metastatic, and rarely curable. Autocrine stimulation of multiple G protein-coupled neuropeptide receptor systems contributes to the transformed growth of SCLC. The ability of neuropeptide receptors to stimulate phospholipase C and mobilize intracellular Ca2+ indicates that Gq family members of heterotrimeric G proteins are a convergence point mediating autocrine signaling by multiple neuropeptides in SCLC. Expression of a GTPase-deficient, constitutive active form of an alpha q family member, alpha 16Q212L, in SCLC markedly inhibited growth of the cells in soft agar and tumor formation in nude mice. SCLC lines expressing alpha 16Q212L exhibited 2-4-fold elevated basal phospholipase C activity, but neuropeptide and hormone-regulated intracellular Ca2+ mobilization was nearly abolished. The data suggest that Ca2+ mobilization is an obligatory signal in neuropeptide-stimulated growth of SCLC. In addition, the proline-directed c-Jun NH2-terminal kinases/stress-activated protein kinases, which are members of the mitogen-activated protein kinase family, were stimulated approximately 2-fold in parental SCLC in response to exogenous neuropeptides and muscarinic agonists and were constitutively activated to the same degree in alpha 16Q212L-expressing SCLC. Thus, alpha 16Q212L expression induced desensitizaton of neuropeptide-stimulated Ca2+ signaling and persistent activation of the c-Jun NH2-terminal kinase/stress-activated protein kinase pathway. We propose that the induction of discordant signaling by selective perturbation of receptor-regulated effector systems leads to the inhibition of SCLC cell growth.

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.

Radiant heat-induced hyperthermia in mice: in vivo effects on the immune system.

Whole-body hyperthermia (WBH) in mice was induced by 2-4-h exposure to radiant heat resulting in core body temperatures of 38.5-40.4 degrees C, and correlated directly with the magnitude and duration of heat treatment. Two-hour heat treatments in this temperature range did not consistently affect generation of antibody-forming cells in vivo, while 4-h treatments at temperatures greater than or equal to 40 degrees C significantly suppressed the antibody-forming cell response. The capacity of lymphocytes from similarly heated mice to generate antibody-forming cells in vitro was not affected, suggesting that the observed in vivo suppression may be mediated by circulating factors rather than by some heat-induced alteration in the cells themselves. In vivo treatment did not alter T-cell responsiveness to the mitogen Con-A or delayed-type hypersensitivity responses to sheep red blood cells. Quantitative and flow cytometric assessment of splenic and thymic lymphocyte numbers showed that WBH did not alter absolute numbers of lymphocytes but did temporarily change the proportions of lymphocyte subsets. An immediate increase in splenic L3T4+ cells was observed, followed within 18 h by an overall decrease in Lyt 2+ and Thy 1.2+ T-cells. In the thymus the percentages of mature T cells increased. In general, only minimal effects of heat on the immune responses of normal mice could be demonstrated.

A randomized study of the efficacy of adjuvant local graft irradiation following renal transplantation.

A prospective randomized study investigating the effectiveness of adjuvant local graft irradiation (LGI) following renal transplantation was performed at Georgetown University Hospital from 1983 until 1988. One hundred and thirty-eight patients were enrolled in the study with 117 patients receiving cadaver kidney transplantations and 21 patients receiving living related kidney transplantations. Seventy-one patients were randomized to receive adjuvant local graft irradiation consisting of 600 cGy in four fractions with chemical immunosuppression whereas the remaining 67 patients received chemical immunosuppression only (control group). The two groups were comparable at entry with respect to potentially important prognostic variables. Median follow-up for all patients was 30 months. The 3-year actuarial allograft success rate was 75% and 68% for the local graft irradiation and control groups, respectively. A nonsignificant trend favoring the irradiated group was noted. Subgroup analysis of the 21 recipients of kidneys from living related donors suggested an improvement in allograft survival for the local graft irradiation arm. Cadaver allograft survival was not significantly different between the two treatment arms. There was no apparent benefit in kidney function or time to the first rejection episode in the group receiving local graft irradiation.

Induction of human lymphokine-activated killer cells by IFN-alpha and IFN-gamma.

By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.

Age-related changes in the degeneracy of the mouse T-cell repertoire.

Aging is associated with a decline in T-cell-dependent immune responses. As the number of T-cells remains relatively constant throughout life, the observed decline in T-cell function may reflect qualitative changes in the T-cells themselves or in the composition of the T-cell population. In this investigation, the quality of the T-cell response was studied in young-adult and aged B6C3F1 mice by assessing the degeneracy of the hapten-specific proliferative response to the antigen-MHC complex. Degeneracy was defined as the ability of lymph node cells from mice contact-sensitized to the hapten TNP to proliferate in response to in vitro stimulation with haptenated allogeneic antigen-presenting cells, thus escaping strict MHC restriction. It was found that, although degeneracy occurred in both age groups, it was more prevalent and of a greater magnitude in the young than in the old lymphocytes. These results suggest that there are qualitative differences in T-cell populations derived from young-adult and aged mice.