Genetic variations in cancer-related significantly mutated genes and lung cancer susceptibility.

BACKGROUND: Cancer initiation and development are driven by key mutations in driver genes. Applying high-throughput sequencing technologies and bioinformatic analyses, The Cancer Genome Atlas (TCGA) project has identified panels of somatic mutations that contributed to the etiology of various cancers. However, there are few studies investigating the germline genetic variations in these significantly mutated genes (SMGs) and lung cancer susceptibility. PATIENTS AND METHODS: We comprehensively evaluated 1655 tagged single nucleotide polymorphisms (SNPs) located in 127 SMGs identified by TCGA, and test their association with lung cancer risk in large-scale case-control study. Functional effect of the validated SNPs, gene mutation frequency and pathways were analyzed. RESULTS: We found 11 SNPs in 8 genes showed consistent association (P < 0.1) and 8 SNPs significantly associated with lung cancer risk (P < 0.05) in both discovery and validation phases. The most significant association was rs10412613 in PPP2R1A, with the minor G allele associated with a decreased risk of lung cancer [odds ratio = 0.91, 95% confidence interval (CI): 0.87-0.96, P = 2.3 × 10-4]. Cumulative analysis of risk score built as a weight sum of the 11 SNPs showed consistently elevated risk with increasing risk score (P for trend = 9.5 × 10-9). In stratified analyses, the association of PPP2R1A:rs10412613 and lung cancer risk appeared stronger among population of younger age at diagnosis and never smokers. The expression quantitative trait loci analysis indicated that rs10412613, rs10804682, rs635469 and rs6742399 genotypes significantly correlated with the expression of PPP2R1A, ATR, SETBP1 and ERBB4, respectively. From TCGA data, expression of the identified genes was significantly different in lung tumors compared with normal tissues, and the genes' highest mutation frequency was found in lung cancers. Integrative pathway analysis indicated the identified genes were mainly involved in AKT/NF-κB regulatory pathway suggesting the underlying biological processes. CONCLUSION: This study revealed novel genetic variants in SMGs associated with lung cancer risk, which might contribute to elucidating the biological network involved in lung cancer development.

Natural antibodies to the human T cell lymphoma virus in patients with cutaneous T cell lymphomas.

Sera from patients with cutaneous T cell lymphoma and leukemia were screened for the presence of natural antibody to the human T cell lymphoma (leukemia) virus, HTLVCR, using a solid-phase radioimmunoassay. Sera from two patients, including patient CR, from whose cultured T lymphoblastic cell line (HUT102), the retrovirus HTLVCR was isolated, reacted specifically with proteins of HTLVCR. Serum from patient CR also reacted specifically with proteins of HTLVMB, an independent but highly related retroviral isolate from a patient with Sezary T cell leukemia. The specificity for HTLVCR proteins was demonstrated by solid-phase immunocompetition assays and competition radioimmunoprecipitation assays. Analysis of radioimmunoprecipitates indicated that the natural antibodies were directed against HTLVCR core proteins with molecular weights of 24,000 and 19,000 (p24 and p19). Whereas the serum reactivities for HTLVCR proteins were shown to be highly specific, additional reactivities seen against proteins of animal retroviruses including GaLV, SSV, FeLV, and BaEV were clearly shown not to be viral specific but rather were due to reactivity with cellular antigens contaminating the viral preparations or with related antigens present in fetal calf serum. These results demonstrating natural antibodies to HTLVCR provide the first evidence for a specific antibody response to a retrovirus in humans.

Clinicopathological and therapeutic significance of CXCL12 expression in lung cancer.

Interactions between CXCL12 and its receptors CXCR4 or CXCR7 are involved in tumor growth and metastasis in various types of human cancer. However, CXCL12 expression and its role in lung cancer are not fully elucidated. Here we examined the expression of CXCL12 in 54 lung cancer cell lines consisting of 23 small cell lung cancers (SCLCs) and 31 non-small cell lung cancers (NSCLCs). CXCL12 was overexpressed in lung cancer cell lines compared to non-malignant human bronchial epithelial cell lines (N = 6). CXCL12 expression was positively but weakly correlated with the expression of CXCR4 or CXCR7. We also examined CXCL12 expression in 89 NSCLC specimens and found that CXCL12 expression was significantly higher in tumor specimens from female patients, non-smokers and adenocarcinoma patients. Small interfering RNAs targeting CXCL12 inhibited cellular proliferation, colony formation and migration of CXCL12-overexpressing lung cancer cells; however, this inhibition did not occur in lung cancer cells that lacked CXCL12. Furthermore, the anti-CXCL12 neutralizing antibody mediated inhibitory effects in three lung cancer cell lines that overexpressed CXCL12, but not in two CXCL12 non-expressing lung cancer cell lines nor two non-malignant bronchial epithelial cell lines. The present study demonstrates that: CXCL12 is concomitantly overexpressed with CXCR4 or CXCR7 in lung cancers; CXCL12 is highly expressed in NSCLCs from females, non-smokers and adenocarcinoma patients; and disruption of CXCL12 inhibits the growth and migration of lung cancer cells. Our findings indicate that CXCL12 is required for tumor growth and provide a rationale for the anti-CXCL12 treatment strategy in lung cancer.

High levels of intracellular bombesin characterize human small-cell lung carcinoma.

"Small cells" or "oat cells" characterize a virulent form of lung cancer and share many biochemical properties with peptide-secreting neurones. The neuropeptide bombesin is present in all small-cell lines examined, but not in other lung cancer cell lines, suggesting that bombesinergic precursor cells in lung may give rise to this disease.

Abnormalities in structure and expression of the human retinoblastoma gene in SCLC.

Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in 90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.

Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23).

A specific, acquired chromosomal abnormality (deletion 3p) has been found in at least one chromosome 3 in 100 percent of the metaphases in 12 of 12 cell lines cultured from human small-cell lung cancer tissue and in 2-day tumor culture specimens from three patients. Analysis of the shortest region of overlap shows the deletion to be 3p(14-23). This specific change was not seen in five of five lung cancer cell lines other than small-cell lung cancer or in two lymphoblastoid lines cultured from cells of small-cell lung cancer patients whose tumors had the 3p deletion.

p53: a frequent target for genetic abnormalities in lung cancer.

Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.

Prevalence and specificity of LKB1 genetic alterations in lung cancers.

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.

c-Jun N-terminal kinase is activated in non-small-cell lung cancer and promotes neoplastic transformation in human bronchial epithelial cells.

c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.

Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification.

DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22,000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia.

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p.

A deletion involving chromosome 3p (14-23) characteristically occurs in small cell lung cancer (SCLC). Reduction to homozygosity, rather than complete loss, is typically observed for genes in the deleted region. Lack of expression for genes encoded by this region, implying inactivation of all alleles, has not been previously described. We have examined the expression of aminoacylase-1 (ACY-1), encoded by chromosome 3p21, using both an electrophoretic activity assay and a monoclonal antibody-based ELISA. A variety of human tissues, including lung, brain, liver, kidney, heart, adrenal medulla, and erythrocytes have previously been tested for ACY-1 activity and antigen; all but erythrocytes are positive. Thus, ACY-1 is expressed in all nucleated human cells examined to date. ACY-1 was undetectable in a significant number of SCLC cell lines (4/29) and tumors (1/8), but not in non-small cell lung cancer (NSCLC) cell lines (0/19) or tumors (0/9), nor in a variety of other human cell lines (0/15) or colon tumors (0/8). In addition, reduced (approximately 10% of normal) ACY-1 expression was common in SCLC cell lines (14/29) and tumors (3/8), but not in NSCLC cell lines (1/19) or tumors (0/9), nor in other human cell lines (0/15) or colon tumors (0/8). Thus, low or undetectable ACY-1 expression is highly specific for SCLC and occurs in both cell lines and tumor tissue. The finding of undetectable ACY-1 expression in SCLC supports the hypothesis that inactivation of all alleles of specific chromosome 3p genes occurs in a SCLC in a fashion analogous to Rb gene inactivation in retinoblastoma, and suggests that the structural gene for ACY-1 may be closely linked to a putative SCLC tumor suppressor gene.

Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer.

The p53 gene initially was thought to be an oncogene, but recent evidence suggests that wild-type p53 can function as a tumor suppressor gene in lung, colon, and breast cancer as well as less common malignancies. This study reports the first identification of intronic point mutations as a mechanism for inactivation of the p53 tumor suppressor gene. Abnormally sized p53 mRNAs found in a small cell and a non-small cell lung cancer cell line were characterized by sequence analysis of cDNA/PCR products, the RNase protection assay and immunoprecipitation. These mRNAs were found to represent aberrant splicing leading to the production of abnormal or no p53 protein. Sequence analysis of genomic DNA revealed that a point mutation at the splice acceptor site in the third intron or the splice donor site in the seventh intron accounts for the abnormal mRNA splicing. In one patient the same intronic point mutation was found in the tumor cell line derived from a bone marrow metastasis and in multiple liver metastases but not in normal DNA, indicating that it occurred as a somatic event before the development of these metastases. These findings further support the role of inactivation of the p53 gene in the pathogenesis of lung cancer and indicate the role of intronic point mutation in this process.

Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer.

Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.

Restriction fragment length polymorphism studies show consistent loss of chromosome 3p alleles in small cell lung cancer patients' tumors.

Previous karyotypic analysis of human small cell lung cancer cell lines has demonstrated a consistent deletion of a portion of the short arm of chromosome 3(p14-23). DNA prepared from tumors and normal tissues obtained from 24 small cell lung cancer and two extrapulmonary small cell cancer patients was hybridized to four probes that detect restriction fragment length polymorphisms within chromosome region 3p14-21. Of the 25 patients who were heterozygous for at least one marker in this region in the DNA from normal tissue, 23 (92%) showed an unequivocal loss of heterozygosity in the DNA from their tumor tissue. From these studies we conclude that loss of alleles from the short arm of chromosome 3 is a consistent finding in unselected small cell lung cancer patients' tumor DNA.

Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines.

myc family oncogene amplification in tumor cell lines established from small cell lung cancer patients and its relationship to clinical status and course.

44 small cell lung cancer cell lines established from 227 patients were studied for myc family DNA amplification (c-myc, N-myc, and L-myc). Two of 19 lines (11%) established from untreated patients' tumors had DNA amplification (one N-myc and one L-myc), compared with 11 of 25 (5 c-myc, 3 N-myc, and 3 L-myc) cell lines (44%) established from relapsed patients' tumors (P = 0.04). The 19 patients who had tumor cell lines established before chemotherapy treatment survived a median of 14 wk compared with 48 wk for the 123 extensive stage patients who did not have cell lines established (P less than 0.001). Relapsed patients whose cell lines had c-myc DNA amplification survived a shorter period (median of 33 wk) than patients whose cell lines did not have c-myc amplification (median of 53 wk; P = 0.04). We conclude that myc family DNA amplification is more common in tumor cell lines established from treated than untreated patients' tumors, and c-myc amplification in treated patients' tumor cell lines is associated with shortened survival.

Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.