Evaluation of phenotypic alteration by microcell-mediated chromosome transfer.
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Animals↗
Biomedical subjects
J D Hunt
Publications and source records attributed to J D Hunt.
Manipulation of the helper T cell response to influence antigenic competition occurring with a multivalent vaccine.
The reduction in antibody observed following inoculation with multiple heterologous Dichelobacter nodosus pili antigens is thought to be due to competition between antigen-specific B cells for a limited amount of T cell help. We demonstrate here that this competition is not further influenced by the expansion of cross-reactive antibody secreting cells at the expense of serogroup specific antibody secreting cells. The T cell determinants of pili recognized by sheep and BALB/c mice have been defined using 15 residue peptides. These T cell determinants include cross-reactive determinants in the conserved amino terminal region of the antigen. Here we investigate the effect of expanding the pili-specific T cell population by priming with pili derived T cell determinants. It was not possible to increase the antibody elicited in response to the multivalent vaccine by priming mice with either a synthetic peptide spanning a T cell determinant or with reduced and alkylated or heterologous serogroups of pili 4 weeks before inoculation with the multivalent vaccine. A strategy designed to increase the T cell population by inoculating animals with pili covalently coupled to an extrinsic T cell determinant was pursued.
Amino Acid Sequence↗
Activity of anti-erbB-2 recombinant toxin OLX-209 on lung cancer cell lines in the absence of erbB-2 gene amplification.
The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.
Adenocarcinoma↗
Effects of anti-erbB-2 (HER-2/neu) recombinant oncotoxin AR209 on human non-small cell lung carcinoma grown orthotopically in athymic nude mice.
The recombinant oncotoxin AR209 [e23(Fv)PE38KDEL; formerly OLX-209] was developed to treat neoplasia that expresses the c-erbB-2 (HER-2/neu) protein product p185(erbB-2). The AR209 compound contains a single-chain antibody domain specific for p185(erbB-2), coupled with a portion of the Pseudomonas exotoxin. The drug has been shown to be effective in inhibiting cells that overexpress erbB-2 due to gene amplification and in cells that do not contain amplified erbB-2 but express slightly higher levels of the protein than normal cells do. To test the efficacy of AR209 on human lung tumors, athymic nude mice were inoculated intrathoracically with a cell line derived from a poorly differentiated lung adenocarcinoma. This cell line, termed 201T, expresses moderately elevated levels of p185(erbB-2) 7.6-fold over normal bronchial epithelium. Mice treated with i.v. injections of AR209 for 5 weeks after orthotopic tumor implantation had smaller tumors and in 20% of cases showed no evidence of disease. The data from this study indicate that AR209 may be an effective treatment for patients with non-small cell lung cancers that express p185(erbB-2).
Animals↗
Immunological parameters associated with antigenic competition in a multivalent footrot vaccine.
A murine model for antigenic competition with multivalent D. nodosus pili vaccine has been established that parallels the phenomenon observed in sheep where levels of antibody, specific for any particular serogroup of pili, are significantly lower following vaccination in the presence of multiple serogroups of pili than with that serogroup alone. This competition was observed in both high and low responder strains of mice and was not dependent on the multiplicity of the antigens in the multivalent vaccine but could be observed with a large excess of a single heterologous serogroup. Competition was manifest by a reduction in the number of serogroup-specific antibody secreting cells elicited in response to vaccination. The antibody response to a single serogroup of pili reached a plateau at high doses and it was at these doses that antigenic competition was most pronounced, under conditions where both B- and T-cell responses were limiting. The limit in T-cell responsiveness was not imposed at the level of presentation of antigen. Pili-specific T cells were largely cross-reactive for different serogroups, and under conditions of limiting T-cell stimulation within a lymph node the available T cells would have to be shared between B cells specific for each serogroup of pili, which may in turn result in the decrease of serogroup-specific antibody induced following inoculation with the multivalent vaccine.
Animals↗
Production of gastrin-releasing peptide by a non-small cell lung carcinoma cell line adapted to serum-free and growth factor-free conditions.
Gastrin-releasing peptide is an important growth-modulating factor in developing lung epithelium. It is known to be produced by small cell carcinomas of the lung, and an autocrine loop involving gastrin-releasing peptide and its receptor has been demonstrated in many small cell lung tumors. We investigated whether such an autocrine loop could also be demonstrated in non-small cell lung carcinoma, since gastrin-releasing peptide is known to stimulate human bronchial epithelial cells, from which non-small cell tumors should emerge. We report here that gastrin-releasing peptide is produced by a bronchiolo-alveolar carcinoma cell line (A549) adapted to serum-free and growth factor-free conditions. A549 cells adapted to these conditions, termed A549-R0 cells, display extensive membrane interdigitations, Golgi apparatus, and secretory-like granules, and grow as a mixture of attached colonies and floating cells. Gastrin-releasing peptide is present in the conditioned medium produced by A549-R0 cells. Colony formation of cells derived from a squamous cell carcinoma of the lung, 239T, was stimulated 9-fold by A549-R0 conditioned medium or by authentic gastrin-releasing peptide, measured in serum-free conditions. The growth stimulatory activity was inhibited by a monoclonal antibody to gastrin-releasing peptide. Transcripts for receptors for the bombesin family of peptides were also demonstrated in A549-R0 cells and 239T cells. These results demonstrate that non-small cell lung carcinomas can secrete gastrin-releasing peptide and can also respond to the peptide.
Base Sequence↗
Cytogenetic analysis of 63 non-small cell lung carcinomas: recurrent chromosome alterations amid frequent and widespread genomic upheaval.
A detailed cytogenetic analysis of 63 non-small cell lung carcinomas (NSCLCs) was carried out for identification of recurrent chromosomal alterations. Most specimens displayed very complex karyotypes with multiple numerical and structural changes (median number, 31). Losses of chromosomes 9 (65% of cases) and 13 (71%) were the most frequent numerical changes. Loss of the Y was often observed in tumors from males. Gain of chromosome 7 was also frequent (41%). Chromosome arms 1p, 1q, 3p, 3q, 6q, 7q, 8q, 9p, 11q, 17p, and 19q were particularly prone to rearrangement. The chromosome arm most often contributing to losses was 9p (79%). Other arms that were frequently lost included 3p, 6q, 8p, 9q, 13q, 17p, 18q, 19p, 21q, 22q, and the short arm of each acrocentric chromosome. The percentage of cases with loss of 3p was significantly higher in squamous cell carcinomas (94%) than in adenocarcinomas (60%). There was also a statistically significant increase in the proportion of cases with gains of 1q, 7p, and 11q in adenocarcinomas compared to squamous cell carcinomas. Several recurrent isochromosomes and unbalanced exchanges were found. Among these was i(5p), which was observed in nine tumors, eight of which displayed adenomatous features. An i(8q) was identified in six cases, including five adenocarcinomas. Double minutes and/or homogeneously staining regions were seen in seven specimens. These data indicate that numerous chromosome alterations contribute to the pathogenesis of NSCLC and that, amid this widespread genomic disarray, recurrent abnormalities exist that could have biological and clinical implications.
Adult↗
Ability to culture resectable non-small cell lung carcinomas is correlated with recurrence.
Present clinicopathologic staging of non-small cell lung cancer is limited in its ability to provide more than a general prognostic estimate in patients with lung cancer who have resectable disease. This study was performed to identify whether the ability to adapt tumor tissue from resectable (stage I to IIIa) non-small cell lung cancer was associated with a poorer prognosis and an increased risk for early tumor recurrence. We attempted to culture a tumor specimen obtained from 90 patients with resectable non-small cell lung cancer. We used a culture medium conditioned by exposure to the lung cancer cell line A549-1, a known producer of autocrine lung cancer growth factors, and provided tumor colony scaffolding using a feeder layer of inactivated fibroblasts, and found these measures improved tumor culture yields. Twenty-two cell lines were obtained, a success rate of 24.4%. Tumor recurrences were more common (79%) among the culture-positive patients than among the culture-negative patients (37.5%; p < 0.002). For all patients, survival at 19 months in the culture-positive patients was 50.0%, compared with 83.6% in the culture-negative patients (p < 0.005). The median survival for the culture-positive patients was 15 months, versus 21.7 months for the culture-negative patients (p < 0.004). The establishment of a culture was a predictor of shortened survival for patients with stage I disease. In patients with stage I disease, survival at 19 months was 54.5% for the culture-positive patients, versus 89% for the culture-negative patients (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)
Carcinoma, Non-Small-Cell Lung↗
Antigenic competition in a multivalent foot rot vaccine.
The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa. Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili. This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA. The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody. Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition. Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili.
Agglutinins↗
Expression of mRNA for three bombesin receptor subtypes in human bronchial epithelial cells.
Gastrin-releasing peptide (GRP), the mammalian form of the amphibian peptide bombesin, may act as a growth-regulatory peptide in the lung. Cultured human bronchial epithelial (HBE) cells have previously been found to proliferate in response to GRP or bombesin. Three receptors have been identified for bombesin and its analogs, namely gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), and bombesin receptor subtype 3 (BRS-3). The human genes for these receptors have been cloned and sequenced. We used the polymerase chain reaction to detect transcripts for these three receptor subtypes in HBE cells cultured from bronchial mucosal biopsies. Primers specific for each receptor subtype were used to amplify DNA fragments from reverse-transcribed mRNA isolated from these cultures. An amplified fragment was detected for GRPR in seven of nine cases, for NMBR in six of 10 cases, and the BRS-3 in three of nine cases. We have also amplified message for NMBR in five of five fresh bronchial biopsies, but message for GRPR could not be detected. In preliminary evaluation of two biopsies, there was also no evidence of BRS-3 expression. Additionally, we have detected transcripts for GRPR and NMBR in four of seven and seven of seven cases, respectively, of cultured non-small cell lung carcinomas (NSCLC). BRS-3 expression was seen in one of seven carcinomas in culture. Identity of the amplified fragments was confirmed by restriction enzyme digestion and Southern blotting. Furthermore, GRP induced expression of the early-response gene, c-jun, in an HBE cell culture and an NSCLC cell line. GRP also promoted colony formation in the NSCLC cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Cytogenetic abnormalities in non-small cell lung carcinoma: similarity of findings in conventional and feeder cell layer cultures.
Primary tumors from 39 patients with non-small cell lung carcinoma (NSCLC) were examined for cytogenetic abnormalities by conventional short-term harvest (1-39 days) of primary cultures of minced solid-tumor tissues and by harvest of monolayer cultures of tumor tissue (6 days to 5 months) on murine fibroblast feeder layers. A successful karyotype was obtained with both methods in nine of 39 cases. Among the remaining 30 cases, a successful karyotype was obtained in eight cases by the conventional method only and in three cases by the feeder cell method only. The success rates were 44% for the conventional method, and 31% for the feeder cell method, and the combined success rate was 51% for one or the other method. The feeder culture method, in which harvests were usually performed at later times than with the conventional method, generally produced metaphases with superior banding, which allowed clearer definition of cytogenetic abnormalities. In addition, cell lines were established in eight of these cases by the feeder cell method. Karyotypes from the longer-term harvests typically were very similar to those from short-term conventional cultures. Minor numerical differences and/or a few additional structural abnormalities were noted in seven of the nine cases analyzed by both methods. Overall, however, even in karyotypes from 5-month cultures, the prominent recurrent changes and modal chromosome numbers observed in short-term cultures were still present. The results indicate that long-term culture with fibroblast feeder cells is a valid means of obtaining cells from solid lung tumors for cytogenetic and molecular analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Transfer and expression of the human interleukin-4 gene in carcinoma and stromal cell lines derived from lung cancer patients.
Introduction of the interleukin-4 (IL-4) gene into cells derived from human tumor tissue provides a means for generating a specific tumor vaccine. Such a vaccine could be produced by either transducing tumor-derived stromal cells with the IL-4 vector and coinjecting tumor cells, or by transducing the tumor cells themselves. We have developed a protocol for culturing cells from non-small cell lung tumors and routinely produce tumor cultures from 25% of tumors, and stromal cultures from > 80% of specimens. Several of these cultures were transduced with the incompetent retroviral vector G1NaSvi4.25, which encodes the human IL-4 cDNA and the G418-resistance gene. Infection of cells by viral titers of 2-5 x 10(4) plaque-forming units/ml, and a multiplicity of infection of 0.1:1 to 1:1 yielded transfer efficiencies of 3.3-32.0 transfectants per 10(4) cells in six of eight attempts. Following selection with the neomycin analog G418, IL-4-producing cells were isolated. IL-4 titers ranged from 142 to 593 U/ml/10(6) in a 24-h collection. Successful transfer of the IL-4 gene was demonstrated by polymerase chain reaction amplification of cDNA derived from reverse-transcribed total RNA, by immunohistochemistry, and by enzyme-linked immunosorbent assay. The IL-4-producing cells were shown to stimulate the proliferation of autologous peripheral blood lymphocytes in one individual by 7.5-fold over control and by 4.1-fold over non-IL-4 producing tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Transforming growth factor-alpha gene expression in late-gestation fetal rat lung.
Transforming growth factor-alpha (TGF-alpha) is a structural homologue of epidermal growth factor and competes for binding on a common transmembrane protein receptor/kinase. Although TGF-alpha appears to be more important than epidermal growth factor in embryogenesis and mammalian organogenesis, there is little information regarding its expression in developing lung. Accordingly, we measured levels of immunoreactive TGF-alpha and its gene expression in late-term fetal rat lung during the transition from the canalicular (19-20 d) to the saccular (21 d) stage. We report that at 19 d gestation intrapulmonary levels of TGF-alpha were 1.4 +/- 0.3 pmol/mg protein as determined by RIA, but had decreased by 50% at 21 d. To determine if the TGF-alpha gene is expressed in lung, RNA isolated from fetal rat lung was reverse transcribed, and a 302-bp fragment corresponding to a portion of the TGF-alpha gene was amplified by polymerase chain reaction. Southern blot hybridization with a 32P-labeled 2.3-kb EcoRI fragment of rat TGF-alpha cDNA clone showed a pattern of declining expression during late gestation. Therefore, fetal rat lung expresses TGF-alpha, as is evidenced by the synthesis of both the message and the protein. Because levels of protein were highest in the period of canalicular lung development when the respiratory acinus is formed and vascularized, a potential role for this intrapulmonary growth factor in pulmonary remodeling is suggested.
Animals↗
Molecular evaluation of abnormalities of the short arm of chromosome 1 in neuroblastoma.
Cytogenetic analyses have documented the consistent deletion of part of the short arm of chromosome 1 in neuroblastoma cells suggesting the presence of a suppressor gene in this chromosomal region. To determine, the smallest region of deletion overlap at the molecular level on independently derived tumors and to define the location of the breakpoints more precisely, Southern analyses were performed on a somatic cell hybrid panel containing the normal and altered chromosomes 1 from seven neuroblastoma lines. By this method we were able to analyze a panel of 20 cloned sequences and two isozymes to determine the location of the breakpoints. Our findings indicate that the proximal breakpoints of chromosome 1 deletions ranged over a distance of more than 50 cM with the most distal deletion breakpoint occurring between MYCL1 and D1S57. In addition, using restriction fragment length polymorphisms, it was determined that in at least three of the five cell lines in which MYCL1 was deleted from a chromosome 1, the gene was translocated to another chromosome thus retaining the diploid complement. We propose that the neuroblastoma susceptibility gene is located distal to MYCL1 and that there is another gene which is linked to MYCL1 that may be involved in this neoplasm.
Adolescent↗
Excision of N-myc from chromosome 2 in human neuroblastoma cells containing amplified N-myc sequences.
Amplification of one of three growth-stimulating myc genes is a common method by which many tumor types gain a proliferative advantage. In metastatic human neuroblastoma, the amplification of the N-myc locus, located on chromosome 2, is a dominant feature of this usually fatal pediatric cancer. Of the many models proposed to explain this amplification, all incorporate as the initial step either disproportionate overreplication of the chromosomal site or recombination across a loop structure. The original locus is retained within the chromosome in the overreplication models but is excised in the recombination models. To test these models, we have used somatic cell hybrids to separate and analyze the chromosomes 2 from a neuroblastoma cell line containing in vivo amplified N-myc. Our results demonstrate that N-myc is excised from one of the chromosomes, suggesting that deletion is a requisite part of gene amplification in a naturally occurring system.
Animals↗
Agarose gel electrophoresis in isozyme separation and visualization.
Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.
Animals↗
Relationship between changes in the histologic subtype of small cell carcinoma of the lung and the response to chemotherapy.
It has been documented that changes in the histopathologic subtype of small cell carcinoma of the lung (SCCL) may occur after chemotherapy. The significance of such changes with respect to response to treatment has not yet been studied. In a retrospective review of 25 patients, we correlated their response from chemotherapy with morphologic changes seen in subsequent histologic material. Eleven patients responded to therapy and 14 failed to respond. A difference in original histologic subtype was found in 10 (71%) of the nonresponders and in only two (18%) of the responders. The difference was statistically significant (p less than 0.05). We conclude that patients with "pure" SCCL in the initial biopsy specimen who fail to respond to chemotherapy are likely to have mixed or combined histologic subtypes in subsequent tissue specimens, although we cannot preclude their pre-existence. An attempt to search for different histologic subtypes is warranted in patients who do not respond to chemotherapy regimens considered to be efficacious in SCCL.
Aged↗
No aggravation of the course of experimental glomerulonephritis in spontaneously hypertensive rats.
Functional and morphologic glomerular alterations induced by antiglomerular basement membrane (anti-GBM) nephritis were investigated in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto controls (WKY) for assessment of the role of systemic hypertension in immunologically mediated renal injury. Over a 6-week period serial measurements of systolic blood pressure (BP), serum creatinine (SCreat), creatinine clearance (CCreat), and urinary albumin excretion (UAlbV) were obtained with inulin clearances (CInulin) at the end of the study. Renal tissue was examined by light microscopy (LM), electron microscopy, immunofluorescence, flash 3H-thymidine autoradiography (AR), and staining for nonspecific esterase (NSE). Immunologic humoral response was evaluated by measurement of rat anti-rabbit IgG antibody production. At all time periods studied, SHR and WKY rats with anti-GBM nephritis demonstrated comparable elevations in SCreat and UAlb V as well as diminution of CCreat and CInulin as compared with non-nephritic control rats of each strain. In nephritic WKY rats mild hypertension developed, whereas in nephritic and control SHR rats marked elevations in BP developed. Morphologic injury as assessed by percent glomerular crescents and hypercellularity on LM, numbers of monocyte macrophages by NSE staining, immunofluorescence for IgG, C3, fibrinogen and Ia positivity, and numbers of glomerular 3H-thymidine-labeled cells by AR was notably comparable in both nephritic strains. Humoral antibody responses were also shown to be similar in all rats studied. These results demonstrate that the 5-week course of experimental anti-GBM nephritis is not exacerbated by systemic hypertension. Glomerular autoregulatory capacity may be important in determining the extent of immune-mediated renal injury.
Albuminuria↗