Lens epithelial cell elongation in the absence of microtubules: evidence for a new effect of colchicine.

Embryonic chick lens epithelial cells cultured in serum-supplemented medium elongated in the absence of microtubules after treatment with the antimicrotubule drug nocodazole. Colchicine, at concentrations lower than those that dissociate microtubules, blocks cell elongation and the associated increase in cell volume. These results indicate that an increase in cell volume, not microtubules, is responsible for lens cell elongation and suggest a previously undescribed effect of colchicine on cell volume regulation.

Antibody diversity.

Three important aspects of immunoglobulin gene organization and structure have emerged from studies of cloned immunoglobulin kappa chain genes. (i) Multiple variable genes are encoded separately in the genome of both immunoglobulin-producing and uncommitted (embryonic) cells, thereby establishing the evolutionary base for generating immunoglobulin diversity. (ii) These genes exist as many small, closely related families (subgroups) that share close sequence homology largely within their own subgroup. (iii) Comparison of two cloned variable gene segments derived from a single subgroup reveals a feature of their structure that distinguishes them from fixed genes (that is, globin genes) and provides, through extensive surrounding sequence homology, a large target for intergenic recombination. This last observation suggests that a simple recombination mechanism may account for their genetic instability in both germ line and somatic cells.

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.

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.

Absence of hereditary p53 mutations in 10 familial leukemia pedigrees.

Germline p53 mutations have been identified in the Li-Fraumeni syndrome but the role of such mutations in familial leukemia is not established. The p53 gene was examined by single-strand conformation polymorphism analysis of exons 4-8 in 10 families with multiple members affected with leukemia. The diagnoses included acute and chronic leukemias and Hodgkin's disease. Identified in two families were p53 mutations that were nonhereditary. These included a 2-bp deletion in exon 6 found in the lymphoblast DNA of one child whose sibling, cousin, and several adult relatives had acute leukemia. The other nonhereditary p53 mutation was a transition at codon 248 (CGG to CAG, arginine to glutamine) found in the lymphoblasts of a patient with a preleukemic syndrome and acute lymphoblastic leukemia (ALL) whose brother is a long-term survivor of ALL. Thus, p53 mutations were found to occur in two families but both were nonhereditary. Moreover, in the remaining eight families no p53 mutation was identified in the regions of p53 where most mutations have been found in other cancers. Although p53 mutations sometimes may be present, they do not appear to be a primary event responsible for hereditary susceptibility to familial leukemia. This study suggests involvement of other genes or mechanisms.

Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia.

The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.

Multiple mechanisms for transcriptional regulation of the myc gene family in small-cell lung cancer.

The molecular mechanisms reported to regulate the expression of myc family genes are multiple and complex and include gene amplification, transcriptional activation, transcriptional attenuation, and mRNA stability. We have investigated which of these mechanisms are responsible for the extreme variation in myc gene family mRNA levels observed in human small-cell lung cancer cell lines. In addition to gene amplification, a block to nascent mRNA chain elongation, causing attenuation of transcription, is an important regulatory mechanism controlling the steady-state levels of c-myc and L-myc mRNA. The loss of transcriptional attenuation is correlated with overexpression of these two genes in cell lines which do not show gene amplification. Expression of c-myc mRNA appears to be dependent on promoter activity and attenuator function. In contrast, regulation of expression of the N-myc gene does not involve transcriptional attenuation; steady-state mRNA levels are correlated with promoter activity as well as gene amplification. We conclude that transcriptional regulation of each member of the myc gene family is accomplished by a different assortment of complex mechanisms, including gene copy number, promoter activation, and transcriptional attenuation. Interference at multiple points in this complex regulatory process appears to be an important mechanism by which small-cell lung cancer and other human tumors evade growth control.

Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties.

We have described the establishment and biochemical characterization of 50 small cell lung carcinoma (SCLC) cell lines. Further analysis of these data, combined with studies of morphology and growth characteristics, indicates that 35 (70%) of the lines retained typical morphology (SCLC, intermediate subtype), growth characteristics (growth as tightly packed floating cellular aggregates, long doubling times and low colony-forming efficiencies), and biochemical profile (presence of L-dopa decarboxylase, bombesin-like immunoreactivity, neuron-specific enolase, and high concentrations of brain isoenzyme of creatine kinase). They are referred to as classic SCLC lines. The remaining 15 (30%) lines had discordant expression of the biochemical markers; they retained high concentrations of brain isozyme of creatine kinase, but had significantly lower concentrations of neuron-specific enolase and lacked L-dopa decarboxylase and bombesin-like immunoreactivity. These cell lines are called variants. SCLC variant lines could further be divided into (a) biochemical variant lines having variant biochemical profile but retaining typical SCLC morphology and growth characteristics; and (b) morphological variant (SCLC-MV) lines having variant biochemical profile, altered morphology (features of large cell undifferentiated carcinoma) and altered growth characteristics (growth as loosely attached floating aggregates, relatively short doubling times and cloning efficiencies). Fifty-five clones derived from the three SCLC subclasses retained their parental phenotypes. In SCLC-MV lines there was a near constant relationship between variant morphology, altered growth characteristics and amplification of the c-myc oncogene; classic SCLC and biochemical variant SCLC lines were not amplified. Variant morphologies frequently are present in SCLC tumors at autopsy, and most SCLC-MV lines reflect changes that had occurred in the tumors from which they were derived. Because SCLC-MV tumors behave more virulently in the patient and are radioresistant in vitro, these findings are of considerable biological and clinical interest.

Additive and differential biological activity of alpha-interferon A, difluoromethylornithine, and their combination on established human lung cancer cell lines.

The effect of human recombinant leukocyte interferon A (IFN-alpha A) and DL-alpha-difluoromethylornithine (DFMO) as single drugs and in combination on the in vitro growth, cell cycle distribution, activity of the enzyme L-dopa decarboxylase, and expression of the c-myc and N-myc oncogenes was studied in human lung cancer cell lines. In vitro growth activities were tested in concentrations ranging from 10 to 50,000 IU/ml for IFN-alpha A and from 0.1 to 10 mM for DFMO by means of the soft agarose clonogenic assay using continuous drug exposure. Ten well established small cell lung cancer (SCLC) cell lines including five cell lines of the classic and five of the variant phenotype, two cell lines derived from adenocarcinoma of the lung, and one large cell lung cancer cell line were included in the study. We found that IFN-alpha A inhibited the growth only of the variant phenotype of SCLC with an approximate drug concentration yielding a 50% inhibition of colony growth of 1000 IU/ml. None of the SCLC classic cell lines was inhibited significantly. The growth inhibition of IFN-alpha A correlated with the proliferation rate of the tumor. IFN-alpha A inhibited one of two adenocarcinoma cell lines and 0 of 1 large cell lung cancer cell line. DFMO inhibited the colony formation of 10 of 10 SCLC cell lines, 2 of 2 adenocarcinoma cell lines, and 0 of 1 large cell lung cancer cell line with a drug concentration yielding a 50% inhibition of colony growth of 1 mM. No difference between the classic and variant phenotypes of SCLC was found. The combination of IFN-alpha A and DFMO resulted in an additive cytostatic effect in all cell lines tested. The same result, i.e., an additive cytostatic effect, was obtained for two SCLC cell lines that were tested in liquid culture. Neither single drugs nor their combination led to an accumulation of cells in a particular phase of the cell cycle nor did it affect the activity of the SCLC classic marker enzyme L-dopa decarboxylase. In addition, IFN-alpha A, DFMO, and their combination did not affect the expression of the c-myc and N-myc oncogenes in cell lines NCI-N417 and NCI-H526, respectively, following 4, 24, and 72 h of continuous drug exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GST)-TRAIL extracellular domain fusion proteins were produced to analyze TRAIL-induced apoptosis. Only GST-TRAIL constructs containing regions homologous to the Fas self-association and ligand binding domains could induce apoptosis. GST-TRAIL induced significant (>90%) apoptosis in just one of eight normal and one of eight malignant breast cell lines. All other lines were relatively resistant to TRAIL-induced apoptosis. Activating TRAIL receptors DR4 and DR5 were expressed in all normal and malignant breast cell lines. The inhibitory receptor TRID was highly expressed in one of four normal and two of seven malignant breast cell lines. DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of cell lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-induced apoptosis in most breast cell lines. By fractional inhibition analysis, the toxicity of the combination of TRAIL and doxorubicin or 5-fluorouracil was synergistic compared with either agent alone. In contrast, melphalan and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and methotrexate did not augment it in any cell line. Augmentation of TRAIL-induced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation. This was evidenced by the fact that chemotherapy agents that synergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocked by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk). The combination of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In contrast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e.g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk. These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. In summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no difference in sensitivity is found between normal and malignant cell lines. Treatment with chemotherapy provides an approach to sensitize breast cancer cells to TRAIL-induced apoptosis.

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.

Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma.

The p53 gene was examined in primary or metastatic tumors from six patients with rhabdomyosarcoma (RMS) and in five RMS cell lines by screening methods including single-strand conformation polymorphism analysis, the RNase protection assay, sequencing of complementary DNA subclones, and Southern blotting. Six original tumors were of embryonal histology, four alveolar, and one mixed. p53 mutations were identified in four of the six tumors or cell lines derived from tumors with embryonal histology and in one of the four with alveolar histology. Consistent with p53 allele loss, each mutation was found in the homo- or hemizygous state. One tumor showed a G to C transversion at p53 codon 213 (arginine to proline), and another showed deletion of the entire gene. The p53 mutations in cell lines included a codon 248 C to T transition (arginine to tryptophan) in RD and a codon 280 A to T transversion (arginine to serine) in RH30. The cell line CTR contained a 4-base pair deletion at codons 219/220 in exon 6 with resultant frame shift and premature termination in exon 7. These data support the role of diverse types of p53 mutations in the pathogenesis and/or progression of a significant proportion of cases of childhood RMS.

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.

Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions.

Accumulating evidence indicates that lung cancer arises due to multiple genetic changes in both dominant oncogenes, such as ras, and tumor suppressor genes, such as p53. In this report we examined whether the wild-type p53 gene is able to suppress in vitro and/or in vivo cellular growth of lung cancer cell lines which carry multiple genetic abnormalities. Introduction of a wild-type p53 complementary DNA expression vector into lung cancer cell lines carrying either a homozygous deletion (NCI-H358) or a missense mutation (NCI-H23) in the p53 gene greatly suppressed tumor cell growth. In contrast, p53 expression vectors bearing lung cancer derived mutations affecting single amino acids had lost this growth suppressing ability.

Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2.

We investigated whether combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trastuzumab could enhance the specific killing of cells that overexpress the erbB-2 receptor. The combination resulted in an enhancement of TRAIL-mediated apoptosis in all cell lines overexpressing erbB-2 receptor compared with either reagent alone. In contrast, there was no effect in cell lines with low levels of the erb-B2 receptor. Trastuzumab treatment resulted in down-regulation of the erbB-2 receptor in all erbB-2-overexpressing cell lines. Similar enhancement of TRAIL toxicity was observed when the erbB-2 receptor was down-regulated using antisense oligodeoxynucleotides. Down-regulation of the erbB-2 receptor protein by trastuzumab or antisense oligodeoxynucleotides decreased Akt kinase activation but not mitogen-activated protein kinase activation. Down-regulation of Akt kinase activity by a phosphatidylinositol 3'-kinase inhibitor (LY294002) also resulted in enhancement of TRAIL-mediated apoptosis. Expression of a constitutively active form of Akt kinase in an erbB-2-overexpressing cell line completely abrogated the increase in TRAIL-mediated apoptosis by trastuzumab and significantly reduced the biological effect of either reagent alone. Therefore, down-regulation of the erbB-2 receptor by trastuzumab enhances TRAIL-mediated apoptosis by inhibiting Akt kinase activity. These data suggest that the combination of trastuzumab and TRAIL may allow enhanced therapeutic efficacy and specificity in the treatment of erbB-2-overexpressing tumors.

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

A novel germline p53 splicing mutation in a pediatric patient with a second malignant neoplasm.

A novel germline p53 splicing mutation was identified in a pediatric patient with two metachronous primary cancers that are constituent tumors of the Li-Fraumeni syndrome. Genomic DNA from the second tumor showed the same mutation and loss of heterozygosity at the p53 locus. The mutant mRNA and protein were present in the tumor tissue. In contrast, in the normal tissues bearing the germline mutation in the heterozygous state, predominantly normal mRNA was expressed and the mutant p53 protein was not detectable. The functional silence and relative lack of mutant p53 mRNA expression in the normal tissues of this patient may be caused by decreased stability or decreased production. If this proves a more general pattern of expression of mutant p53 in individuals with germline mutations, these findings may explain the paucity of tumors in individuals affected with the Li-Fraumeni syndrome.

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.

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.