Analysis of p16 and p21(Cip1) expression in tumorigenic human bronchial epithelial cells induced by asbestos.

Although asbestos is carcinogenic to humans, the mechanism(s) by which it induces cancer is unknown. Using tumor cell lines generated previously by asbestos treatment of immortalized human bronchial epithelial (BEP2D) cells, we examined alterations in p16 and p21(Cip1) genes together with their protein levels. Results were compared with untreated BEP2D cells, normal human bronchial epithelial cells (NHBE), as well as non-tumorigenic fusion cell lines generated by fusing tumor cells with BEP2D cells. No deletion in the p16 gene was found in any of the tumor cell lines examined. Although p16 protein was expressed at a similar level in tumor and BEP2D cells, there was a fourfold decrease in its expression among NHBE cells. In contrast, both the protein and mRNA expression levels of p21(Cip1) were decreased by about threefold in tumor cell lines when compared with either BEP2D or NHBE cells, which had a similar expression level. Expression of p21(Cip1) mRNA was restored to the control level in all the fusion cell lines examined. The results suggested that down regulation of p21(Cip1) expression is linked to the tumorigenic conversion of BEP2D cells by asbestos.     

Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA

An in vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human exocervical epithelial cells is described. Eight HPV-immortalized human exocervical epithelial cell lines were established; all the lines contained either integrated HPV16 or 18 sequences and expressed HPV mRNAs. Thus, integration and expression appear to be required for immortalization. Immortalized cells (greater than 200 population doublings to date) divided rapidly (doubling time of 30 to 46 h) and morphologically resembled primary cultures of normal human exocervical epithelial cells. They expressed a keratin pattern consistent with their origin from exocervical epithelium. When cultured at high density or in the presence of serum they terminally differentiated. Sublines resistant to terminal differentiation were selected by growth in serum-supplemented medium. Keratin pattern changes suggest they have some properties in common with cervical squamous carcinoma cells. However, HPV-immortalized cell lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcinogenic by itself. These cell lines represent an appropriate model for studying factors that regulate HPV gene expression in normal cervical epithelial cells and examining the influence of cocarcinogens on neoplastic progression.     

Optical detection and grading of lung neoplasia by Raman microspectroscopy

The aim of this study was to investigate whether Raman spectroscopy could be used to identify and potentially grade lung neoplasia in cell samples. Normal human bronchial epithelial cells (HBEpCs) were analyzed by Raman spectroscopy and compared with (i) HBEpCs expressing human papillomavirus (HPV) type 16 E7 or CDK4; (ii) the immortalized bronchial epithelial cell line BEP2D and (iii) its asbestos-transformed derivative AsbTB2A. Overall, Raman spectroscopy, in combination with a linear discriminant analysis algorithm, was able to identify abnormal cells with a sensitivity of 91% and a specificity of 75%. Subdivision of the cell types into 3 groups, representing normal cells (HBEpCs), cells with extended lifespan (HBEpCs expressing HPV 16 E7 or CDK4) and immortalized/transformed cells (BEP2D and AsbTB2A) showed that Raman spectroscopy identifies cells in these categories correctly with sensitivities of 75, 79 and 87%, and specificities of 91, 85 and 96%, respectively. In conclusion, Raman spectroscopy can, with high sensitivity, detect the presence of neoplastic development in lung cells and identify the stage of this development accurately, suggesting that this minimally invasive optical technology has potential for lung cancer diagnosis.     

Growth Dependence of Human Papillomavirus 16 DNA-positive Cervical Cancer Cell Lines and Human Papillomavirus 16-Transformed Human and Rat Cells on the Viral Oncoproteins

The dependence on human papillomavirus (HPV) Oncoproteins of the growth of cervical cancer cell lines [C4-1, HeLa (both containing HPV 18 DNA), CaSki and SiHa (both containing HPV 16 DNA)], HPV 16-transformed human embryonic kidney cells, and HPV 16-transformed rat brain and 3Y1 cells was examined by using antisense RNA approaches. The cells were transfected with plasmids expressing RNA antisense to the HPV 16 or 18 open reading frames E6E7, together with plasmids expressing the hygromycin B resistance gene, and drug-resistant colonies were scored three weeks later. In all the human cell lines, the efficiency of colony formation was lowered by RNA antisense to the resident HPV type. Some of the rat cell lines responded to the antisense plasmids, but some did not. From a nonresponding rat tumor line (3Y1HP-1T), cell clones with various levels of E7 protein were isolated after transaction with the antisense plasmid, and were examined for anchorageindependent growth in soft agar. The colonies formed by the clones with lower E7 levels tended to be smaller and fewer than those formed by the clones with higher E7 levels. These findings strongly suggest that some of the transformed or cancer phenotypes of cells in vitro are dependent, even after extensive passages and malignant changes, on expression of the oncoproteins of the resident HPV.     

Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNAs exhibit dysplastic differentiation in vivo.

Human papillomavirus (HPV) DNAs are detected in approximately 90% of anogenital carcinomas. To assess directly the effect of HPV on squamous differentiation, normal human cervical and foreskin epithelial cells and cells immortalized by recombinant HPV DNAs were transplanted beneath a skin-muscle flap in athymic mice. Xenografts containing normal cells formed well-differentiated stratified squamous epithelia 2 to 3 weeks after transplantation, but cell lines immortalized by four HPV types (HPV16, HPV18, HPV31, and HPV33) detected in anogenital cancer exhibited dysplastic morphology and molecular alterations in gene expression characteristic of intraepithelial neoplasia. Morphological alterations were accompanied by delayed commitment to terminal differentiation, alterations in the pattern of involucrin expression, and reductions in levels of involucrin and keratin 1 RNAs. HPV18-immortalized cells developed dysplastic changes more rapidly than cells immortalized by HPV16 DNA. These results show that human genital epithelial cells immortalized by HPV DNAs detected in genital cancers undergo dysplastic differentiation in vivo.     

Radiation-induced deletion of chromosomal regions containing tumor suppressor genes in human bronchial epithelial cells

While several specific genetic alterations have been associatedwith malignant transformation of human bronchial epithelialcells, they are not all present in every tumor and there isreason to believe that additional genes important for loss ofreplication control in these cells remain to be identified.In an effort to develop a human bronchial epithelial cell modelsuitable for identification and functional analysis of genesinvolved in loss of replication control, and for studying thegenetic basis of the multi-stage phenotypic changes associatedwith tumorigenesis, we treated multiple independent populationsof the human papillomavirus (HPV)-immortalized human bronchialepithelial cell line BEP2D with ionizing radiation. Followingirradiation, cell lines representing the radiated populationswere established from single soft agar-selected colonies. Theselines—R2B5SA, R3B5DSA, R2H9S, R2H16S, R2H18S and R3D7S—werecompared cytogenetically to the parent cell line and found tohave new chromosomal deletions involving putative or confirmedtumor suppressor genes, including chromosome 13 in most R2B5SAcells and all R3B5SA cells, chromosomes 11p and 22 in R216S,and 3p, 11p and 22 in R2H18S. The R2B5SA cells that have lostone copy of chromosome 13 overgrow the culture but are not tumorigenic,suggesting that loss of a single copy of chromosome 13 confersgrowth advantage but not tumorigenicity. The data confirm thationizing radiation induces many large chromosomal alterationsincluding chromosomal loss, translocation and deletion and thatfollowing radiation it is possible to isolate immortalized non-tumorigeniccell lines monosomic for regions known or suspected to containtumor suppressor genes. The cell lines described here providepowerful models for further investigation of putative tumorsuppressor genes including identification, functional analysisand stage of transformation at which they are operative.     

E6/E7 genes of human papilloma virus type 18 induced immortalization of human fetal esophageal epithelium

To study the role played by human papilloma virus (HPV) in carcinogenesis, immortalized esophageal epithelial cells were induced by E6 and E7 genes of HPV type 18 and the biological behavior was studied. Human fetal esophageal epithelial cells were transfected with recombined HPV18E6E7AAV and were cultured and passaged in medium M199. In both the 10th passage (SHEE10) and the 31st passage (SHEE31), their proliferative rates by flow cytometry and their abilities to grow and form colonies in soft agar, or to form tumors in SCID mice were examined. The HPV18 genes of E6E7 and its expression were determined using PCR methods. Cellular telomerase activity was detected by TRAP and chromosomes were analyzed by standard method. Immortalized cell lines of esophageal epithelium induced by the HPV18E6E7 were successfully established and cultured for >100 passages over 4 years. The result of PCR showed that the E6E7 gene of HPV18 was detectable in both cell clones. Both of them were unable to grow in soft-agarose medium and failed to produce tumors in SCID mice. Flow cytometry demonstrated an average of 43% proliferation index in SHEE31, but 28% in SHEE10. Telomerase activity was clearly identified in SHEE31 but not in SHEE10. Cytogenetic analysis demonstrated progression of chromosomal abnormalities with increasing trisome. Our data indicated that genes E6/E7 of the HPV18 were capable of inducing immortalization in fetal esophageal epithelial cells. The immortal phenotype requires both activation of telomerase and genetic alterations that abrogate normal differentiation and promote cellular proliferation. This cell line can assist us to characterize the role played by HPV in carcinogenesis.     

Immortalization of human adult prostatic adenocarcinoma cells by human papilloma virus HPV16 and — 18 DNA

Primary prostate epithelial and prostate adenocarcinoma cells cultured in serum-free medium grew for up to 10 passages before senescence. Cells from prostate adenocarcinoma of a 55-year-old patient without lymph node involvement were transfected with plasmids containing recombinant human papilloma virus HPV16 or HPV18 DNA and the selectable neomycinresistance gene. After G-418 selection, cells underwent crisis, and surviving cells infected with retroviruses encoding the HPV18 E6/E7 genes (HPV-PAC1), transfected with a head-to-tail dimer of the complete HPV16 genome (HPV-PAC2), or transfected with HPV18 E6/E7 early genes (HPV-PAC3) were established. HPV-PAC1 and HPV-PAC2 cultures appeared morphologically similar to primary cultures even after 40 passages. However, HPV-PAC2 cultures had a clonal morphology. All lines were positive for cytokeratin 18, had acquired vimentin expression, and contained either HPV16 or HPV18 sequences integrated into host DNA. None was tumorigenic in nude mice or formed colonies in soft agar. These cells did not secrete prostate specific antigen nor respond to androgen although tamoxifen inhibited the growth of the cells. Immunohistochemistry showed no evidence of p53 overexpression. Further characterization of these cell lines and examination of their response to chemotherapeutic agents may provide relevant information for the study of hormone-independent PC.     

Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential.

The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.     

Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection.

Human papillomavirus (HPV) DNAs are detected in most genital dysplasias and cancers, suggesting that these viruses perturb epithelial growth and differentiation. The E6 and E7 genes of HPV type 18 induce immortality in keratinocytes cultured from genital tract epithelia, and the immortal cell lines display aberrant squamous differentiation. To examine whether the E6 and E7 proteins directly alter keratinocyte growth and differentiation, high-titer recombinant retroviruses were constructed for efficient transfer and expression of HPV-18 genes E6, E6* and E7 in cultures of normal human keratinocytes. Infection with retroviruses encoding E6 and E7 stimulated cell proliferation, reduced the requirement for bovine pituitary extract and induced immortality. E6 and E7 also delayed but did not prevent the onset of terminal squamous differentiation. The magnitude of effects on growth and differentiation of cultured cells was directly related to levels of E7 protein expression. Thus, expression of the HPV-18 E6 and E7 genes stimulates cell proliferation and delays differentiation of keratinocytes in vitro.     

Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells: implication for mechanism

Although exposure to asbestos fibers is associated with the development of lung cancer, the underlying mechanism(s) remains unclear. Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, we previously showed that UICC chrysotiles can malignantly transform these cells in a stepwise fashion before they become tumorigenic in nude mice. In the present study we used cDNA expression arrays to screen differentially expressed genes among the tumorigenic cells. A total of 15 genes were identified, 11 of which were further confirmed by northern blot. Expression levels of these genes were then determined among transformed BEP2D cells at different stages of the neoplastic process, including non-tumorigenic cells that were resistant to serum-induced terminal differentiation, early and late passage transformed BEP2D cells, five representative tumor cell lines and fused tumorigenic-control cell lines which were no longer tumorigenic. A consistent 2- to 3-fold down-regulation of the DCC (deleted in colon cancer), Ku70 and heat shock protein 27 genes were detected in all the independently generated tumor cell lines while expression levels in early transformants as well as in the fusion cell lines remained normal. In contrast, all the tumor cell lines examined demonstrated 2- to 4-fold overexpression of the insulin receptor and its signal transduction genes. Differential expression of these genes was completely restored in the fusion cell lines examined. No alteration in c-jun or EGF receptor expression was found in any of the cell lines. Our data suggest that activation of the insulin receptor pathway and inactivation of DCC and Ku70 may cooperate in malignant transformation of BEP2D cells induced by asbestos.     

High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.     

Localization of tumor suppressor gene candidates by cytogenetic and short tandem repeat analyses in tumorigenic human bronchial epithelial cells

Radon exposure is associated with increased risk for bronchogenic carcinoma. Mutagenesis analyses have revealed that radon induces mostly multi-locus chromosome deletions. Based on these findings, it was hypothesized that deletion analysis of multiple radon-induced malignant transformants would reveal common mutations in chromosomal regions containing tumor suppressor genes responsible for malignant transformation. This hypothesis was supported by a previous study in which tumorigenic derivatives of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were established following irradiation with 30 cGy of high linear energy transfer radon-simulated alpha-particles. Herein, we describe the analyses of 10 additional tumorigenic derivative cell lines resulting from the irradiation of five additional independent BEP2D populations. The new transformants have common cytogenetic changes, including the loss of chromosome (ch)Y, one of three copies of ch8, one of two copies of ch11p15-pter and one of three copies of ch14. These changes are the same as those reported previously. Analysis of PCR-amplified short tandem repeats of informative loci confirmed the loss of heterozygosity (LOH) at 12 loci spanning the length of ch8 in cell lines from four of the total of eight irradiation treatments to date and the loss of chY in all cell lines (8 of 8). LOH analysis with a total of 17 informative loci confirmed loss on ch14 in transformants from seven of eight irradiation treatments and indicated a 0.5-1.7 cM region of common involvement centered around locus D14S306. No LOH was detected at any of the informative loci on ch11. The overall results support our stated hypothesis. Further studies are currently in progress to determine whether the ch8 and ch14 regions contain genes with tumor suppressor function in bronchial epithelial cells.     

Effect on cancer cells of plasmids that express antisense RNA of human papillomavirus type 18.

Some human squamous cell carcinomas contain DNA of human papillomaviruses (HPV) and express RNA from the E6 and E7 genes. We have examined the effect of plasmids that express antisense RNA of these genes on the growth of the human cancer cell lines HeLa, C4-1, and 1483, which contain HPV type 18 DNA. As controls, the human cancer cell line 183 and the Vero line of monkey kidney cells were used, which do not contain HPV. Plasmids were introduced into the cells by electroporation; cells that contained HPV type 18 accepted the antisense-expressing plasmids at a lower frequency than the cells that lacked HPV. Cell lines were developed from HeLa cells that contained sense- or antisense-expressing plasmids, and lines that contained antisense-expressing plasmids showed slower growth, reduced ability to form colonies in soft agar, and increased serum requirements. The use of antisense HPV RNA might be a suitable approach to gene therapy of HPV-expressing human cancers.     

Smad7基因过表达的促增殖作用机制探讨

目的 研究Smad7过表达使人支气管上皮细胞系增殖能力增强的机制。方法 用Smad7真核表达载体PCISmad7．neo同携带有报告基因碱性磷酸酶的c-myc顺式增强子元件pMyc-SEAP共转染,检测Smad7基因对增殖信号通路的影响。用RT-PCR方法,检测稳定转染Smad7基因前后永生化及恶性化的人支气管上皮细胞系BEP2D和BERF35T2细胞内c-myc、p15、p21表达水平的变化,调查Smad7基因对TGF-β介导的抗增殖基因反应的调控。结果 报告基因检测结果表明,Smad7基因可使参与增殖信号通路的c-myc顺式增强子元件活性增强。RT-PCR结果表明,在稳定转染Smad7基因的细胞中,c-myc表达上调,p15表达降低,对TGV-β刺激的应答丧失,p21表达降低。结论 Smad7基因可通过调控TGF-β介导的抗增殖基因应答来影响细胞的生长、增殖能力。     

Role of oncogenes and tumour suppressor genes in human lung carcinogenesis.

Six families of activated protooncogenes, ras, raf, fur, neu, jun and myc have so far been associated with human lung cancer. Human bronchial epithelial cells in vitro are being used to investigate the functional role of these specific oncogenes and growth regulatory genes in carcinogenesis and tumour progression. When transferred into normal human bronchial epithelial cells by the highly efficient protoplast fusion method, the v-Ha-ras oncogene initiates a cascade of events leading to decreased responsiveness of these cells to inducers of squamous differentiation, aneuploidy and, less frequently, 'immortality' and tumorigenicity with metastasis in athymic nude mice. Transfection of the SV40 T antigen gene results in nontumorigenic cell lines that have a nearly normal pathway of terminal squamous differentiation and can be transformed into malignant cells by transfected Ha-ras, N-ras or Ki-ras. The combination of transfected c-myc and c-raf-1 also transforms human bronchial epithelial cells into neoplastic cells that exhibit some phenotypic traits found in small-cell carcinomas. These and other results indicate that proto-oncogenes dysregulate the pathways of growth and differentiation of human bronchial epithelial cells and play an important role in human carcinogenesis. Analyses of allelic deletion and somatic cell hybrids are being used to identify the chromosomal localization of tumour suppressor genes. We have examined 54 non-small-cell bronchogenic carcinomas with 13 polymorphic markers. Loss of heterozygosity was more frequent than among 23 squamous-cell carcinomas than among 23 adenocarcinomas or eight large-cell carcinomas. Loss of heterozygosity for chromosome 17p was found in 89% of cases of squamous-cell carcinoma and 18% of adenocarcinomas. Analysis of chromosome 11 for allelic deletions revealed two commonly deleted regions (11p13 and 11p15.5). Somatic cell hybrids between normal human bronchial epithelial cells and Hut292DM, a lung carcinoma cell line, had a finite lifespan in vitro and were nontumorigenic in athymic nude mice. Tumour suppressive effects of individual or combinations of specific human chromosomes on Hut292DM are being examined by formation of microcell-cell hybrids. Chromosome 11 has tumour suppressor activity in these hybrids. Both of these studies suggest that tumour suppressor genes play a dominant role in lung carcinogenesis and provide in-vitro model systems for isolating these genes by subtraction library and insertional mutagenesis techniques.