Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells.

Cultured bronchial epithelial and fibroblastic cells from humans were used to study DNA damage and toxicity caused by formaldehyde. Formaldehyde caused the formation of cross-links between DNA and proteins, caused single-strand breaks in DNA, and inhibited the resealing of single-strand breaks produced by ionizing radiation. Formaldehyde also inhibited the unscheduled DNA synthesis that occurs after exposure of cells to ultraviolet irradiation or to benzo[a]pyrene diolexpoxide but at doses substantially higher than those required to inhibit the resealing of x-ray-induced single-strand breaks. Therefore, formaldehyde could exert its mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.

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.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.

Replicative epithelial cell cultures from normal human prostate gland.

Epithelial cell cultures of the normal human prostate gland were established. The subculturing of these cultures was accomplished with a novel nonenzymatic technique. These cultures were defined as normal epithelial cells on the basis of ultrastructure, karyotype, and inability to grow in soft agar.

Alpha-particle-induced p53 protein expression in a rat lung epithelial cell strain.

Other investigators have shown that both sparsely ionizing and UV radiation cause cell cycle arrest that is associated with increased expression of wild-type p53 protein. The effect of exposure to alpha-particles from 238Pu on the induction of the p53 protein has now been examined in cultured lung epithelial cells derived from male F344 rats. The number of cells having increased levels of p53 protein was determined by flow cytometry after the cells had been stained with a monoclonal antibody to p53. alpha-Particle irradiation caused a dose-dependent increase in p53 protein levels detectable at doses as low as 0.6 cGy, with no evidence of a threshold. An increase in p53 protein also occurred in X-irradiated cells. However, no increase was seen in cells exposed to less than 10 cGy of X-rays, indicating the existence of a relatively higher DNA damage threshold for sparsely ionizing radiation. In addition, more cells exposed to low doses of alpha radiation had increased p53 protein levels than would be predicted based on the number of nuclei expected to be traversed by an alpha-particle, suggesting that alpha-particles cause genetic damage by mechanisms in addition to direct interactions with DNA.

Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells.

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on 10 human lung carcinoma cell lines were compared to those seen on normal human bronchial epithelial (NHBE) cells. TPA (0.1 to 100 nM) did not enhance the clonal growth rate for any of the cell lines. As little as 3 nM TPA induced the NHBE cells to undergo terminal squamous differentiation and thus completely inhibited their proliferation; in contrast, none of the carcinoma cell lines was significantly inhibited at this concentration, and they all continued to proliferate in as much as 100 nM TPA. To determine if this lack of TPA inhibition of clonal growth reflected resistance to TPA induction of terminal squamous differentiation, we measured the ability of TPA to induce cross-linked envelope formation and to increase plasminogen activator activity in four carcinoma cell lines. Cross-linked envelopes were not induced in two lines, and only a small number were induced in the other two lines relative to NHBE cells; plasminogen activator activity was induced in NHBE cells but not in any of the cell lines.

Chromosomal analysis of human prostatic adenocarcinoma cell lines.

Although detailed cytogenetic analysis has been carried out in many types of cancer, there is little information on the chromosomal makeup of prostatic cancer cells. Karyological analyses of cell lines derived from both metastatic and primary prostatic carcinoma have been carried out by Q-, C-, and sequential banding techniques. The metastatic line, PC-3, isolated from a bone marrow specimen, is an established epithelial line which is tumorigenic in nude, athymic mice and forms colonies in semisolid agar suspension. A subline, PC-3/M, was isolated from a PC-3-induced mouse tumor. Karyotypic analysis of PC-3 by Q- and C-banding showed the cells to be aneuploid at all culture passage levels. The modal chromosome number shifted from 62 to 55 between the 5th and 50th passages. PC-3 has a unique karyotype. Chromosomes 2, 3, 5, 15, and Y were always absent. At least 11 different marker chromosomes were observed. The subline, PC-3/M, had a similar karyotype and retained the parental PC-3 markers. PC-3/M had a more restricted chromosomal frequency distribution range. Nearly 73% of the PC-3/M cells examined had 60 or 61 chromosomes in contrast to the wide distribution seen in PC-3. Silver staining for nucleolus organizer regions indicated that the number of functional nucleolus organizer regions in PC-3 was proportional to the number of acrocentric chromosomes. Banding analysis of PC-5-PI isolated from primary prostatic adenocarcinoma indicated that this line also had a characteristic karyotype with 28% pseudodiploid and 72% pseudotetraploid components. All metaphases examined were partially trisomic in chromosome 9 and lacked a demonstrable Y chromosome.

Differential control by platelet factors of squamous differentiation in normal and malignant human bronchial epithelial cells.

Recently, we developed a nutritionally optimal medium for rapid clonal growth (greater than 1 population doubling/day) of normal human bronchial epithelial (NHBE) cells. Adding fetal bovine or adult human blood-derived serum to this medium depresses the clonal growth rate of NHBE cells in a dose-dependent fashion. In contrast, 10 representative lines of human lung carcinomas either replicate poorly or fail to grow at all when inoculated at clonal density in serum-free medium, and their rates of multiplication increase in direct proportion to the amount of blood-divided serum added to the optimized medium. Thus, the growth factor requirements of these lung carcinoma cell lines are significantly different from those of their normal counterparts. Blood-divided serum reduces the clonal growth rate of NHBE cells by specifically inducing the normal cells, but not lung carcinoma cells, to undergo squamous differentiation. The differentiation-inducing activity was found in platelet lysates. In addition, a growth-inhibiting activity that did not induce squamous differentiation of NHBE cells was also identified in partially purified commercial preparations of platelet-derived growth factor. This observation was in marked contrast to results using human bronchial fibroblasts and human lung carcinoma cell lines; the growth rate of the former was significantly stimulated by commercial preparations of platelet-derived growth factor, whereas the growth rates of the tumor cell lines were unaffected. These results indicate that an aberration in the cellular differentiation as assayed in vitro is positively correlated with cancer and suggests that decreased responsiveness to inducer(s) of differentiation may be a major aspect of bronchial cell carcinogenesis.

Clonal growth of epithelial cells from normal adult human bronchus.

Normal primary epithelial cell cultures devoid of fibroblastic cells have been developed from tissue explants of adult human bronchi. Conditions for clonal growth of secondary cultures of bronchial epithelial cells were optimized by coculturing the human cells with mitomycin C growth-arrested Swiss 3T3 mouse feeder cells, lowering the calcium concentration of medium M199, and supplementing it with hydrocortisone, insulin, cholera toxin, epidermal growth factor, and 1.25% fetal bovine serum. The epithelial cells grew for an average of 35 population doublings and had the normal human karyotype, expressed keratin and blood group antigen epithelial cell markers, metabolized benzo(a)pyrene, and were capable of differentiating into both ciliated and squamous cells. This culture system makes it potentially possible to investigate various aspects of differentiation and carcinogenesis in human bronchial epithelial cells.

Effects of serum, transforming growth factor type beta, or 12-O-tetradecanoyl-phorbol-13-acetate on ionized cytosolic calcium concentration in normal and transformed human bronchial epithelial cells.

Fluctuations in ionized cytosolic calcium ([Ca2+]i) are considered important signals for induction of growth or differentiation in mammalian cells. The resting concentrations of [Ca2+]i in normal and adenovirus 12-SV40 hybrid virus-transformed (BEAS-2B) human bronchial epithelial cells were 63 +/- 15 nM (SD) and 44 +/- 15 nM, respectively. Eight % calcium-free fetal bovine serum rapidly caused a significant increase in [Ca2+]i, while causing both cell types to undergo squamous differentiation. When treated with 8% calcium-free fetal bovine serum, a serum-sensitive subclone of BEAS-2B cells exhibited a higher elevation of [Ca2+]i than a serum-resistant (i.e., not stimulated to differentiate by serum) subclone. However, a serum component involved in the induction of squamous differentiation, transforming growth factor type beta, did not increase [Ca2+]i in either normal cells or BEAS-2B cells. 12-O-Tetradecanoyl-phorbol-13-acetate, an exogenous inducer of squamous differentiation and activator of protein kinase C, did not increase [Ca2+]i, but did attenuate serum-induced elevation of [Ca2+]i. These results suggest that while an increase in [Ca2+]i is associated with serum-induced squamous differentiation, a cytosolic ionized calcium signal is not required for the initiation of the squamous differentiation pathway induced by either transforming growth factor type beta or 12-O-tetradecanoylphorbol-13-acetate.

Effects of formaldehyde, acetaldehyde, benzoyl peroxide, and hydrogen peroxide on cultured normal human bronchial epithelial cells.

The effects of several aldehydes and peroxides on growth and differentiation of normal human bronchial epithelial cells were studied. Cells were exposed to formaldehyde, acetaldehyde, benzoyl peroxide (BPO), or hydrogen peroxide (HPO). The effect of each agent on the following parameters was measured: (a) clonal growth rate; (b) squamous differentiation; (c) DNA damage; (d) ornithine decarboxylase activity; (e) nucleic acid synthesis; (f) aryl hydrocarbon hydroxylase activity; and (g) arachidonic acid and choline release. None of the agents were mitogenic, and their effects were assessed at concentrations which reduced growth rate (population doublings per day) to 50% of control. The 50% of control concentrations for the 6-h exposure were found to be 0.065 mM BPO, 0.21 mM formaldehyde, 1.2 mM HPO, and 30 mM acetaldehyde. BPO-exposed cells were smaller than controls (median cell planar area, 620 sq microns versus 1150 sq microns), and acetaldehyde-exposed cells were larger than controls (median cell planar area, 3200 sq microns). All agents increased the formation of cross-linked envelopes and depressed RNA synthesis more than DNA synthesis. HPO caused DNA single-strand breaks, while formaldehyde and BPO caused detectable amounts of both single-strand breaks and DNA-protein cross-links. Other effects included increased arachidonic acid and choline release due to HPO. The similarities and differences of the effects of these aldehydes and peroxides to those caused by tumor promoters are discussed.

Development of tumorigenicity in simian virus 40-immortalized human bronchial epithelial cell lines.

Of five SV40-transformed clonal human bronchial epithelial cell lines previously shown to be nontumorigenic at early passages (R. R. Reddel et al., Cancer Res., 48: 1904-1909, 1988), two lines (BES-1A1 and BEAS-2B) from different donors have become weakly tumorigenic with further passaging. BES-1A1 passage 26 cells formed tumors in 3 of 9 athymic nude mice given s.c. injections, whereas BEAS-2B cells of > or = 32 passages formed highly cystic tumors at 8 of 58 injection sites after long latency periods [17 +/- 7 (SD) weeks]. These tumors took a total of 36 +/- 8 weeks to reach a diameter of 1.0 cm. Tumor cell lines were established from four BEAS-2B tumors, and these are resistant to the growth-inhibitory effects of serum, an inducer of squamous differentiation in BEAS-2B and normal bronchial epithelial cells. This finding supports the hypothesis that development of resistance to inducers of terminal squamous differentiation may be a step in the process of bronchial carcinogenesis. One of these tumor cell lines, B39-TL, is significantly more tumorigenic than the others and has a deletion from the short arm of chromosome 3 as has been described previously for some naturally occurring human bronchial carcinomas. Thus, from the clonally derived BEAS-2B cell line, cell populations with various degrees of tumorigenicity have developed. Analysis of the changes in these cells may yield insights into the multiple events involved in acquisition of the tumorigenic phenotype.

Comparison of production of transforming growth factor-beta and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines.

Steady state mRNA levels for transforming growth factor beta, platelet-derived growth factor (PDGF) A-chain, and PDGF B-chain were measured in normal human mesothelial cells, SV40 large T-antigen expressing human mesothelial cells, and human mesothelioma cell lines. The mRNA expression level for transforming growth factor beta was similar in all three types of culture while normal human mesothelial cells secrete more transforming growth factor beta than do mesothelioma cell lines or T-antigen expressing mesothelial cells. In contrast, both PDGF A- and B-chain mRNAs are expressed at higher levels in mesothelioma cell lines than in normal human mesothelial cells. PDGF-like mitogenic activity was readily detectable in medium conditioned by a mesothelioma cell line and was undetectable in conditioned medium from normal cells. These results suggest the hypothesis that PDGF may be an autocrine growth factor in mesothelioma.

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Comparison of benzo(a)pyrene metabolism in bronchus, esophagus, colon, and duodenum from the same individual.

The metabolism of benzo(a)pyrene has been investigated in cultured normal human bronchus, colon, duodenum, and esophagus obtained from the same patient. The highest total metabolism was found in bronchus and duodenum, while the highest mean binding level was observed in the bronchus followed, in order, by the esophagus, duodenum, and transverse colon. A 30-fold interindividual variation in the binding level was found in each of the four organs studied, and a positive correlation between the binding levels in bronchus, colon, and duodenum was found. In human bronchus, a positive correlation was found between level of binding of benzo(a)pyrene to DNA and the amount of both benzo(a)pyrene 7,8-diol and the combined group of 3-hydroxybenzo(a)pyrene, benzo(a)pyrene 9,10-diol, and water-soluble metabolites. A significantly higher relative amount of benzo(a)pyrene tetrols and benzo(a)pyrene 9,10-diol was formed by human bronchus compared to the gastrointestinal tissues, while a higher level of benzo(a)pyrene phenols was formed by the latter. The relative distribution of benzo(a)pyrene-DNA adducts was similar in all four organs, the major DNA adduct being formed by trans-addition of anti-7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene to the 2-amino group at guanine. These results indicate that the metabolism of benzo(a)pyrene by at least four different organs is qualitatively similar but that quantitative differences exist.

Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.

Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.

A simple method for generating full length cDNA from low abundance partial genomic clones.

BACKGROUND: PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes. RESULTS: The first approach we used for the isolation of full-length cDNA from two known genomic clones of Hox genes was based on fusion PCR. Here we describe a simple and efficient method of amplification for homeobox D13 (HOXD13) full length cDNA from two partial genomic clones. Specific 5' and 3' untranslated region (UTR) primer pairs and website program (primer3_www.cgv0.2) were key steps involved in this process. CONCLUSIONS: We have devised a simple, rapid and easy method for generating cDNA clone from genomic sequences. The full length HOXD13 clone (1.1 kb) generated with this technique was confirmed by sequence analysis. This simple approach can be utilized to generate full-length cDNA clones from available partial genomic sequences.

Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells.

Human MutT homologue (hMTH1) mRNA was overexpressed in SV-40-transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B cells) and in 11 out of 12 human lung cancer cell lines relative to normal human bronchial epithelial cells. Expression levels of hMTH1 mRNA were inversely proportional to cellular levels of 8-oxo-deoxyguanosine. Together, these results suggest that hMTH1 gene expression may represent a molecular marker of oxidative stress that could ultimately be used to elucidate the temporal relationships between oxidative stress, genomic instability and the development of lung cancer.

Frequency of trisomy 20 in nonmalignant bronchial epithelium from lung cancer patients and cancer-free former uranium miners and smokers.

Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.

Detection of trisomy 7 in nonmalignant bronchial epithelium from lung cancer patients and individuals at risk for lung cancer.

Early identification and subsequent intervention are needed to decrease the high mortality rate associated with lung cancer. The examination of bronchial epithelium for genetic changes could be a valuable approach to identify individuals at greatest risk. The purpose of this investigation was to assay cells recovered from nonmalignant bronchial epithelium by fluorescence in situ hybridization for trisomy of chromosome 7, an alteration common in non-small cell lung cancer. Bronchial epithelium was collected during bronchoscopy from 16 cigarette smokers undergoing clinical evaluation for possible lung cancer and from seven individuals with a prior history of underground uranium mining. Normal bronchial epithelium was obtained from individuals without a prior history of smoking (never smokers). Bronchial cells were collected from a segmental bronchus in up to four different lung lobes for cytology and tissue culture. Twelve of 16 smokers were diagnosed with lung cancer. Cytological changes found in bronchial epithelium included squamous metaplasia, hyperplasia, and atypical glandular cells. These changes were present in 33, 12, and 47% of sites from lung cancer patients, smokers, and former uranium miners, respectively. Less than 10% of cells recovered from the diagnostic brush had cytological changes, and in several cases, these changes were present within different lobes from the same patient. Background frequencies for trisomy 7 were 1.4 +/- 0.3% in bronchial epithelial cells from never smokers. Eighteen of 42 bronchial sites from lung cancer patients showed significantly elevated frequencies of trisomy 7 compared to never smoker controls. Six of the sites positive for trisomy 7 also contained cytological abnormalities. Trisomy 7 was found in six of seven patients diagnosed with squamous cell carcinoma, one of one patient with adenosquamous cell carcinoma, but in only one of four patients with adenocarcinoma. A significant increase in trisomy 7 frequency was detected in cytologically normal bronchial epithelium collected from four sites in one cancer-free smoker, whereas epithelium from the other smokers did not contain this chromosome abnormality. Finally, trisomy 7 was observed in almost half of the former uranium miners; three of seven sites positive for trisomy 7 also exhibited hyperplasia. Two of the former uranium miners who were positive for trisomy 7 developed squamous cell carcinoma 2 years after collection of bronchial cells. To determine whether the increased frequency of trisomy 7 reflects generalized aneuploidy or specific chromosomal duplication, a subgroup of samples was evaluated for trisomy of chromosome 2; the frequency was not elevated in any of the cases as compared with controls. The studies described in this report are the first to detect and quantify the presence of trisomy 7 in subjects at risk for lung cancer. These results also demonstrate the ability to detect genetic changes in cytologically normal cells, suggesting that molecular analyses may enhance the power for detecting premalignant changes in bronchial epithelium in high-risk individuals.