Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

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.

Methylthioadenosine phosphorylase deficiency in human non-small cell lung cancers.

Methylthioadenosine (MeSAdo) phosphorylase, a purine metabolic enzyme, is present in all normal mammalian tissues. A deficiency of this enzyme has been reported in some human leukemias and lymphomas and in a few solid tumors. In the present study, a specific immunoassay was used to assess the enzyme levels in human non-small cell lung cancer cell lines and primary tumors. We also tested the effects of MeSAdo phosphorylase-selective chemotherapy on the in vitro growth of enzyme-positive and enzyme-negative lung cancer cell lines. Of 29 non-small cell lung cancers, 9 (6 cell lines and 3 primary tumors, 31%) lacked detectable immunoreactive enzyme protein. Both 5,10-dideazatetrahydrofolate, an inhibitor of de novo purine synthesis, and methionine depletion, combined with MeSAdo, prevented the growth of the enzyme-negative non-small cell lung cancer cells under conditions in which enzyme-positive cells utilized MeSAdo to endogenously synthesize purine nucleotides and methionine. Our data suggest that MeSAdo phosphorylase deficiency is frequently found in non-small cell lung cancers and can be exploited in designing enzyme-selective chemotherapy.

Homozygous loss of the interferon genes defines the critical region on 9p that is deleted in lung cancers.

Cytogenetic analyses of non-small cell lung cancer have revealed deletions of the short arm of chromosome 9 with breakpoints at 9p11-pter in a significant proportion of tumors. Recent evidence suggests that homozygous loss of the interferon (IFN) and methylthioadenosine phosphorylase (MTAP) genes located on 9p and a tumor suppressor gene closely linked to them is associated with acute lymphoblastic leukemia and with gliomas. We have observed alterations of DNA sequences on 9p which include the IFN genes at a significant frequency in all types of human lung cancers (20 of 56 or 36%). The genetic alterations observed include homozygous or hemizygous deletions of the IFN genes as well as rearrangement of contiguous DNA sequences. In addition to these genomic alterations, 10 of 22 (45%) cell lines examined lacked MTAP enzyme activity. Overall, 24 of 56 (43%) lung cancer cell lines examined had hemizygous or homozygous loss of DNA sequences which include the IFN or MTAP genes. These findings suggest that the putative tumor suppressor gene at this locus contributes to the malignant process in lung cancers, as well as other types of human cancer.

A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity.

Small cell lung cancer (SCLC) tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of a 'tumor suppressor' gene within that region whose functional inactivation may be involved in tumorigenesis. Recently, a hybrid, HA(3)BB9F, was identified that contains a small fragment of human chromosome 3 of approximately 2 Mb on a mouse (A9) background (Killary et. al., 1992). This hybrid was utilized to define a functional tumor suppressor gene within 3p22-p21 which could suppress the tumorigenic properties of the mouse fibrosarcoma cell line. The existence of a tumor suppressor gene in the region 3p22-p21 is supported by the present report which describes the assessment of 89 SCLC and 32 non-SCLC lung cancer tumors and cell lines for the existence of a homozygous deletion(s) at 43 loci on the short arm of chromosome 3. One of the SCLC cell lines was found to harbor a homozygous deletion involving the loss of five markers on chromosome 3p. All five of the markers map to the region 3p21.3-p21.2 and four of the five markers are located within the chromosome 3 fragment exhibiting properties of tumor suppression in the HA(3)BB9F hybrid. The other tumors analysed all retained at least one copy of each of the markers assessed.

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.

High frequency of somatically acquired p53 mutations in small-cell lung cancer cell lines and tumors.

We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Abnormalities of p53 were found in 16/16 cell lines (100%) and in 16/20 tumors (80%). In the SCLC cell lines, mutations (59% missense, 18% nonsense and 23% splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these data that: (1) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events.

Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Lung Cancer Study Group.

The p53 gene has been implicated as a tumor suppressor gene with mutations found in common human cancers. We examined 51 early stage, primary, resected non-small cell lung cancer specimens using an RNAase protection assay and cDNA sequencing. Mutations changing the p53 coding sequence were found in 23/51 (45%) tumor specimens, but not in the corresponding normal lung, were distributed between codons 132 to 283, and included tumors with and without 17p allele loss. Fifteen of the 23 mutations lay in the predicted binding regions for SV40 large T antigen, and 14 were located in regions highly conserved between species. G to T transversions were a common result of p53 mutations in lung cancer compared to other cancers suggesting exposure to different mutagens. In univariate and multivariate analysis the presence of p53 mutations was associated with younger age and squamous histology. However, the presence of p53 mutations was not significantly associated with tumor stage, nodal status or sex and was found in all histologic types of lung cancer. We conclude that somatic mutations in the p53 gene play an important role in the pathogenesis of early stage non-small cell lung cancer.

A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer.

We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of lung cancer. Our PCR-aided assay shows that in most lung-cancer-derived cell lines the amount of D8 transcripts is only 2% or less of that in normal lung tissue. The virtual absence of expression may imply some role of the gene in the development of lung cancer.

Frequent involvement of chromosome 3p alterations in lung carcinogenesis: allelotypes of 215 established cell lines at six chromosome 3p loci.

We have determined the allelotypes of 215 established lung cancer cell lines by PCR analysis at six loci on the short arm of chromosome 3 (3p): D3S3 (3p12-p13), D3S30 (3p13), D3S2 (3p14-p21.1), D3S32 (3p21), D3F15S2 (3p21), and THRB (3p24). Eighty-seven small cell lung cancer (SCLC), 93 non-small cell lung cancer (NSCLC), 6 extrapulmonary SCLC, 6 mesothelioma, and 23 normal B lymphocyte (BL) cell lines were analyzed. Low levels of heterozygosity at all six 3p loci were seen in both the SCLC and NSCLC cells. SCLC cell lines exhibited the lowest frequencies of heterozygosity at D3S3 (3%), D3S2 (3%), D3F15S2 (10%), and THRB (6%) when compared with frequencies of 8, 42, 48, and 34% at these same loci in the normal population. The lowest frequencies of heterozygosities among the NSCLC cell lines were seen at D3S3 (5%), DF15S2 (17%), and THRB (15%). Adenocarcinoma (Ad) was the only subtype of NSCLC that exhibited any heterozygosity (7%) at D3S3. In addition to D3S3, the lowest frequencies of heterozygosity were seen at D3F15S2 for Ad (9%), D3S2 for large cell carcinomas (8%), and THRB for adenosquamous (0%), bronchioloalveolar (0%), and large cell (8%) carcinomas. In summary, the 3p chromosome region near the D3S3 locus (3p12-p13) appears to be involved in all forms of lung cancer with additional involvement of regions close to the D3S2 (3p14-p21.1), D3F15S2 (3p21), and THRB (3p24) loci.

Specific integration of recombinant proviral sequences in ecotropic Gross virus-accelerated AKR thymomas.

Integration and amplification of ecotropic and recombinant proviral sequences in high-molecular-weight cellular DNAs from ecotropic Gross virus-accelerated AKR thymomas were analyzed using an ecotropic-specific probe, p400, and an envelope-specific probe, pAKV-5. New ecotropic proviral sequences were detected at three sites in the DNAs from eight Gross virus-accelerated thymomas following EcoRI restriction endonuclease digestion and at six sites following PvuII restriction endonuclease digestion. The integration of these new ecotropic proviral sequences appeared to be random. Recombinant 3' proviral-cellular DNA junction fragments were detected at 30 sites following digestion with EcoRI. These new recombinant fragments ranged in size from 9.0 to 2.5 kb with 6/8 thymoma DNAs containing a fragment of 2.7 kb. PvuII generated new recombinant 3' proviral-cellular junction fragments that ranged in size from 12.5 to 2.1 kb with 5/8 thymoma DNAs containing a fragment of 2.5 kb. It appears that the leukemia-accelerating ecotropic Gross virus is responsible for the generation of a unique 3' recombinant proviral-cellular junction fragment. This fragment can be detected against a background of randomly integrated ecotropic and recombinant proviruses.

SMX-1 virus-induced inhibition of ecotropic and recombinant proviral sequence amplification in thymuses of AKR mice.

SMX-1 virus delays the appearance of spontaneous thymomas in AKR mice which have been inoculated as young adults by the intrathymic route. Analyses of high-molecular-weight thymus DNAs from SMX-1 virus-inoculated AKR mice indicated the absence of 3' recombinant proviral-cellular DNA junction fragments generated by EcoRI and PvuII digestion. An average of five recombinant proviral fragments were detected in DNAs from spontaneous thymomas that developed in medium-injected control mice. Preleukemic mice that amplify murine leukemia virus-related antigens on their thymocyte surface contained unintegrated proviruses in their thymus DNAs, and 2.3-kilobase EcoRI and 2.1-kilobase PvuII recombinant DNA fragments were detected.

Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts.

A 96,000-dalton glycoprotein, p(96), was present in cell extracts obtained from gs-chf- chicken embryo fibroblasts infected with the avian RNA tumor viruses Rous-associated virus-2 subgroup B (RAV-2) and the Schmidt-Ruppin strain of Rous sarcoma virus subgroup A (SR-RSV-A), as well as from uninfected gsLchf+ (HE) cell extracts. It was not found in cell extracts from uninfected gs-chf- or gs+chf+ (HH) cells, nor from gs-chf- cells infected with envelope-deficient Bryan high-titer Rous sarcoma virus. Immunoprecipitation, kinetic, and biochemical data indicate the this polyprotein contains information that gives rise to the major virion glycoprotein gp85. A second polyprotein of 80,000 daltons, p/80), is also present in the RAV-2- and SR-RSV-A-infected gs-chf- cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicate that p(80) may be an immature form of p(96).

Leukemogenic activity of murine type C viruses after long-term passage in vitro.

Cloned stocks of several murine leukemia viruses (MuLVs) were shown to be leukemogenic for susceptible mice after more than nine years of in vitro passaging in mouse embryo fibroblasts. Tissue culture-grown Rauscher (R-) MuLVs injected into newborn or young adult BALB/c mice induced lymphocytic leukemias in 100% of the animals beginning 80 days post-inoculation. No erythroblastic leukemia was observed even after passaging the tissue-culture-grown R-MuLVs twice through mice, indicating that the component responsible for that disease had been lost or attenuated during growth in fibroblasts. The tissue-culture-grown stock of Moloney (M-) MuLVs likewise induced lymphocytic leukemias in 94% of injected newborn BALB/c mice, and the tissue culture-grown Gross (G-) MuLVs induced lymphocytic leukemias in 42% of injected newborn C3Hf mice. The host range and neutralization characteristics of viruses recovered from animals that became leukemic after injection with the tissue-culture-maintained MuLVs were found to be identical with those of the injected viruses. These data implicate the injected MuLVs in the induction of the leukemias and suggest that the capacity to induce the disease is stably inherited as part of the viral genome even in the absence of expression.

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses IV. Biochemical Typing of Murine Viral Proteins.

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.

Homozygous deletion, rearrangement and hypermethylation implicate chromosome region 3p14.3-3p21.3 in sporadic breast-cancer development.

DNAs from 19 malignant human breast tumors and 2 benign fibroadenomas were analyzed for heterozygosity at 5 polymorphic loci on the short arm of chromosome 3. One homozygous deletion and one rearrangement were identified using probe D3S2 which maps to 3p14.3-3p21.1. This probe also detected novel hybridizing fragments of 2.0 kb and/or 3.4 kb in 6/18 (33%) of the malignant tumor samples that hybridized with the D3S2 probe following digestion with the 5'-methylcytosine-insensitive enzyme MspI. Comparisons of HpaII and MspI digestion showed that all but one of the tumor DNAs analyzed were hypermethylated. The two fibroadenoma DNAs were not as highly methylated and had hybridizing fragments of 3.4 kb after HpaII digestion. These malignant breast-tumor DNAs exhibit 3 mechanisms by which a tumor-suppressor gene hypothesized to reside at 3p14-3p21 could be inactivated: homozygous deletion, rearrangement and hypermethylation, and strongly implicate this 3p chromosome region in breast-tumor development.

Two regions of homozygosity on chromosome 3p in squamous cell carcinoma of the head and neck: comparison with cytogenetic analysis.

BACKGROUND: Loss effecting the short arm of chromosome 3 occurs in nearly 60% of squamous cell carcinomas of the head and neck (SCCHN). Karyotype analysis indicated that these losses occur in two regions, 3p13-p14 and 3p21-p24. To test these findings, we examined tumor DNA from 38 SCCHN cell lines for heterozygosity and homozygosity at 6 polymorphic loci spanning this region. METHODS: The polymerase chain reaction (PCR) was used to amplify polymorphic restriction sites on 3p, the amplified products were digested with the appropriate restriction enzyme, electrophoresed on agarose gels, and assessed for the presence of one or both alleles. The 38 SCCHN cell lines were established from 31 patients and included 16 that had been karyotyped. In 6 cases two or three tumor cell lines established from separate tumors in the same patients were studied. RESULTS: The cell lines exhibited a very low frequency of heterozygosity for the regions 3p12-3p21 (D3S3, D3S30 and D3S2) and distal 3p21-3p24 (D3F15S2 and THRB), when compared with that observed in the normal population. In contrast, D3S32, located within 3p21, was heterozygous in 38% of the tumors which is close to the frequency seen in the normal population (50%). In most cases the PCR results were consistent with the cytogenetic predictions. However, in 4 cell lines 3p loss was predicted from the karyotype, but heterozygosity for D3S32 was present. CONCLUSIONS: These experiments support cytogenetic data that indicate two regions of 3p loss in SCCHN tumors. The 3p regions that show a high frequency of homozygosity may contain tumor suppressor genes involved in the development and/or progression of squamous cancer. The region surrounding D3S32 may contain an essential gene that is conserved in two copies even when much of 3p is lost.

Most sequence differences between the genomes of the Akv virus and a leukemogenic Gross A virus passaged in vitro are located near the 3' terminus.

The 70S genomic RNA of nonleukemogenic AKR(Akv) virus was compared to that of an in vitro passaged, cloned, leukemogenic Gross A virus by fingerprint and sequence analysis. Fifty-seven of the large ribonuclease T1-resistant oligonucleotides of each virus have the same electrophoretic mobility and sequence. Thirteen large ribonuclease T1 oligo nucleotides are unique to the Gross A virus, whereas five are unique to Akv. Four of the oligonucleotides unique to each virus are related by one or two simple base changes. Five of the differences in oligonucleotides are located in the region of the genome that codes for the gag and pol genes. Eight of the differences are located near the 3' poly(A) terminus of the virus. The origins and biological consequences of these differences are discussed.

Presence of murine leukemia virus envelope proteins gp70 and p15(E) in a common polyprotein of infected cells.

The murine leukemia virus envelope proteins, p15(E) and gp70, exhibit a mode of processing distinct from that of virion core proteins according to three criteria. First, the incorporation of both p15(E) and gp70 into virions is more sensitive to the metabolic analogue 2-deoxy-D-glucose than the incorporation of core proteins. Second, the kinetics with which the newly synthesized envelope proteins appear in the released virions is delayed relative to the appearance of core proteins. Third, immunoprecipitation of large polypeptides from infected cells reveals the presence of gp70 and p15(E) in a common precursor distinct from the core polyprotein.