Immune cell cooperation, viruses, and antibodies to nucleic acids in new zealand mice.

The development of autoimmunity in New Zealand mice is related to genetic, immunologic, and viral factors. Evidence is presented to suggest that thymus-dependent immune functions may be depressed and bone marrow-dependent functions augmented in these mice. Antibodies to RNA and DNA appear spontaneously and can also be induced by treatment with rI.rC. Antibodies binding rI.rC-(14)C in human lupus sera, in NZB/NZW F(1) (B/W) mice developing lupus, and in NZB, ALN, and ALN/NZB mice have greatest specificity for reovirus double-stranded RNA. Treatment of B/W mice with RNA and cyclophosphamide induces immunologic tolerance, and suppresses antibodies binding rI.rC-(14)C. During recovery, the specificity of the antibodies is unaltered. Induction of tolerance in this way prevents the accelerated formation of anti-RNA antibodies normally induced by MLV. This finding suggests that virus-accelerated and natural disease occur through a similar mechanism, and supports the hypothesis that viruses may act as antigenic stimuli for a genetically hyper-responsive antibody-producing system.

Should we continue to use the term non-small-cell lung cancer?

Until recently the major clinical question was 'Is it small-cell or non small-cell cancer'. However, advances in conventional and targeted therapy have completely changed the landscape. Identification of the major non-small-cell lung cancer (NSCLC) types (adenocarcinoma and squamous carcinoma) are important for a number of predictive and prognostic reasons, including pemetrexed treatment, anti-angiogenic therapy and administration of tyrosine kinase inhibitors. Fortunately, advances in pathology of lung cancer have kept abreast, with newer, simplified methods to identify the major NSCLC types in small diagnostic samples, and modifications of the pathological classification of adenocarcinomas reflecting changing clinical and molecular concepts. For the patient to obtain maximum benefit from the recent developments in therapeutics, a multidisciplinary approach is required with co-operation between oncologists, surgeons, radiologists and pathologists.

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.

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.

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.

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

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.

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.

Chromogranin A expression in normal and malignant human tissues.

We used a recombinant cDNA probe for human chromogranin A to measure the expression of mRNA encoded by this gene in a variety of normal human tissues and tumor specimens using Northern blot and in situ hybridization analysis. With few exceptions, the expression of chromogranin A mRNA appears to be restricted to normal tissues and tumors of neuroendocrine lineage. However, we have detected mRNA expression of this gene in 1 of 14 cell lines and 2 of 13 tumor specimens of colon adenocarcinoma. The finding of chromogranin A expression in some colon carcinomas suggests that a previously unrecognized subgroup of these tumors has neuroendocrine features. The detection of this subgroup demonstrates the potential for improving tumor classification through the use of techniques and reagents developed by recombinant DNA technology.

Human creatine kinase-B complementary DNA. Nucleotide sequence, gene expression in lung cancer, and chromosomal assignment to two distinct loci.

Using a small cell lung cancer (SCLC) cDNA library, we obtained clones for the creatine kinase-B (CK-B) gene and determined the nucleotide sequence for the protein coding and 3' untranslated region (3' UT). The human translated protein spans 381 residues and the amino acid homology with rabbit CK-B is greater than 98%. We have demonstrated that a nucleic acid probe encompassing the protein coding region will also hybridize to CK-M sequences while a probe derived from the 3' UT region is CK-B specific. When a B-isoenzyme specific sequence is hybridized to Eco RI cut genomic DNA, two independent restriction fragment polymorphisms are detected. We have subsequently localized these two CK-B homologous sequences to chromosomes 14q32 and 16. Finally, we show that increased levels of CK-B seen in SCLC are not accompanied by gene amplification or rearrangement, but reflect a greatly enhanced level of CK-B specific mRNA that is not seen in non-SCLC lines thus far examined.

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.

DNA content analysis by flow cytometry and cytogenetic analysis in mycosis fungoides and Sézary syndrome.

Flow cytometric (FCM) analysis of DNA content was performed on 82 lymph node and peripheral blood specimens from 46 patients with mycosis fungoides and the Sézary syndrome. Overall, 32 of the 46 patients (70%) had aneuploidy detected by FCM. Aneuploidy was present in 63% of the patients at the time of diagnosis before systemic therapy. In these patients, aneuploidy was frequently detected in blood and lymph node specimens scored as negative by cytology and histology, suggesting that unsuspected extracutaneous dissemination is present in many patients at the time of diagnosis. Direct comparison with Giemsa-banded cytogenetic studies showed an excellent correlation of FCM results and cytogenetic chromosome number. However, FCM frequently detected a larger fraction of aneuploid cells, and mitogen-stimulation studies suggest this is the result of preferential stimulation of normal lymphocytes by phytohemagglutinin. Thus, mitogens with a preference for malignant T cells, such as staphylococcal protein A, should be used for cytogenetic analysis of malignant T-cell disorders. At diagnosis, some histologically positive specimens contained only diploid cells by FCM and cytogenetic analysis. These patients had a more indolent clinical course than patients with aneuploidy. Aneuploidy was detected by FCM as either wide G(1) or as discrete aneuploid peaks. The presence of aneuploidy at any time in the clinical course implied a poor prognosis. Discrete hyperdiploid peaks were associated with large cell histology, early relapse, and aggressive clinical course. The development of hyperdiploidy at relapse was documented in four patients and was associated with a transition to large cell histology and a poor prognosis. Similar studies may elucidate differences in natural history and mechanism for transition in histology in other lymphomas and solid tumors.

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.

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.

Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis.

Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.

Complex translocation disrupts c-myc regulation in a human plasma cell myeloma.

A complex translocation has interrupted the third exon of the c-myc gene in human plasma cell myeloma tumor cells and a derivative cell line (NCI-H929). As a result of this rearrangement, a chimeric mRNA is expressed which commences 5' of the c-myc coding region and includes sequences introduced by the translocation event. All of the detectable c-myc-containing mRNA in the tumor and cell line was derived from this rearranged c-myc allele. This chimeric c-myc mRNA, in which most of the germ line c-myc 3' untranslated region has been replaced, was greater than sevenfold more stable than c-myc transcripts with intact 3' ends. This suggests that the 3' untranslated region may play an important role in c-myc mRNA stability.

Cloned fusion product from a rare t(15;19)(q13.2;p13.1) inhibit S phase in vitro.

BACKGROUND: Somatically acquired chromosomal translocation is a common mechanism of oncogene activation in many haematopoietic tumours and sarcomas. However, very few recurrent chromosomal translocations have been reported in more common epithelial tumours such as lung carcinomas. METHODS: We established a cell line HCC2429 from an aggressive, metastatic lung cancer arising in a young, non-smoking woman, demonstrating a t(15;19)(q13.2;p13.1). The breakpoints on chromosomes 15 and 19 were cloned using long distance inverse PCR and we determined by RT-PCR that the translocation results in a novel fusion transcript in which the 3' end Brd4 on chromosome 19p is fused to the 5' end of NUT on chromosome 15q. RESULTS: In total, 128 lung cancer tissues were screened using fluorescent in situ hybridisation, but none of the tumours screened demonstrated t(15;19), suggesting that this translocation is not commonly found in lung cancer. Consistent with previous literature, ectopic expression of wild type Brd4 was shown to inhibit G(1) to S progression. However, we also found that the Brd4-NUT fusion augments the inhibition of progression to S phase compared with wild type Brd4. CONCLUSION: Alteration in cell cycle kinetics is important in tumorigenesis, although the exact role of Brd4-NUT fusion protein in the pathogenesis of lung cancers remains unclear and need to be further elucidated.

Studies on a transplantable murine rhabdomyosarcoma.

Well-differentiated rhabdomyosarcomas developed in 8 of 10 BALB/c mice inoculated with cell-free extracts of two lesions arising in bats previously inoculated with the Moloney strain of murine sarcoma virus. The tumors could be transplanted in BALB/c mice and other strains of mice compatible at the H-2 locus. A tumorigenic clonal cell culture line (R2) was established from a transplanted tumor. Ultrastructural studies of the primary and transplanted tumors and R2 cells revealed thick and thin myofilaments characteristic of myoblasts. These cells contained numerous cisternal type-A virus-like particles, but no type-C particles were found. Attempts to recover, rescue, or chemically induce transforming or nontransforming viruses from transplantable tumors and the R2 cell line were unsuccessful. The tumor cells lacked the murine type-C virus gs antigen. Hybridization data confirmed the apparent lack of viral RNA in the tumor cells.