Normal Human Diploid Fibroblasts Research Articles

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 microM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 microM AG126 (an inhibitor of ERK1/2), but not by 25 microM SP600125 (an inhibitor of JNK1/2) or 5 microM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1-->S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1-->S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.

Suppression of survival in human SKBR3 breast carcinoma in response to metal–chelator complexes is preferential for copper–dithiocarbamate

Since diethyl dithiocarbamate (DEDTC) forms complexes with either zinc or copper, and 8-hydroxyquinoline (8-OHQ) also complexes with copper, we now compared the cytotoxic activity of Cu[DEDTC] 2, Zn[DEDTC] 2 and Cu[8-OHQ] 2. This report shows that at nanomolar levels, only copper–[DEDTC] 2, suppresses proliferation and clonogenicity of SKBR3 human breast carcinoma, concurrently with induction of apoptosis-associated PARP fragmentation. Susceptibility to these agents was paralleled by reactive oxygen generation (ROS) and greater expression of anti-oxidant enzymes like MnSOD and catalase, with no comparable effect on Cu/Zn superoxide dismutase. The lethal effects of Cu[DEDTC] 2 manifested when adding the two separate aqueous components or the preformed synthetic complexes in DMSO, was prevented by N-acetyl cysteine or glutathione, with no comparable protection afforded by non-thiol anti-oxidants like mannitol or DMSO. Exogenously added catalase also protected cells from Cu[DEDTC] 2, suggesting that this complex may kill after the levels of superoxide anion [O 2 −] dismutated by MnSOD increase hydrogen peroxide-related stress. Cu[DEDTC] 2 also induced p21WAF1, a cdk inhibitor usually not inducible in mutant p53 tumors like SKBR3 carcinoma, correlating with dephosphorylation of the Sp1 transcription factor. Concentrations of Cu[DEDTC] 2 cytotoxic for SKBR3 carcinoma did not induce comparable damage versus normal diploid human WI-38 fibroblasts. In contrast to the cytotoxic effect of nM levels of Cu[DEDTC] 2 against SKBRR3 cells, no response was seen in the same cells exposed to 20 μM cis-platin. Since neither DEDTC bound to zinc, nor copper bound to 8-OHQ showed comparable cytotoxicity, our results suggest that the greater activity of copper–DEDTC reflects a specific structure–activity relationship for the active complex. Since Cu[DEDTC] 2 shows more effectiveness than other metal–chelator complexes, it may be worth further investigation as an alternative to cancer therapies.

Bystander effect: Biological endpoints and microarray analysis

In cell populations exposed to ionizing radiation, the biological effects occur in a much larger proportion of cells than are estimated to be traversed by radiation. It has been suggested that irradiated cells are capable of providing signals to the neighboring unirradiated cells resulting in damage to these cells. This phenomenon is termed the bystander effect. The bystander effect induces persistent, long-term, transmissible changes that result in delayed death and neoplastic transformation. Because the bystander effect is relevant to carcinogenesis, it could have significant implications for risk estimation for radiation exposure. The nature of the bystander effect signal and how it impacts the unirradiated cells remains to be elucidated. Examination of the changes in gene expression could provide clues to understanding the bystander effect and could define the signaling pathways involved in sustaining damage to these cells. The microarray technology serves as a tool to gain insight into the molecular pathways leading to bystander effect. Using medium from irradiated normal human diploid lung fibroblasts as a model system we examined gene expression alterations in bystander cells. The microarray data revealed that the radiation-induced gene expression profile in irradiated cells is different from unirradiated bystander cells suggesting that the pathways leading to biological effects in the bystander cells are different from the directly irradiated cells. The genes known to be responsive to ionizing radiation were observed in irradiated cells. Several genes were upregulated in cells receiving media from irradiated cells. Surprisingly no genes were found to be downregulated in these cells. A number of genes belonging to extracellular signaling, growth factors and several receptors were identified in bystander cells. Interestingly 15 genes involved in the cell communication processes were found to be upregulated. The induction of receptors and the cell communication processes in bystander cells receiving media from irradiated cells supports the active involvement of these processes in inducing bystander effect.

Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

Monochloramine (NH 2Cl) is one of the inflammation-derived oxidants, and has various effects on cell cycle, apoptosis and signal transduction. We studied the effects of NH 2Cl on DNA repair response induced by ultraviolet B (UVB) irradiation in normal human diploid fibroblasts, TIG-1. TIG-1 irradiated with 20 mJ/cm 2 UVB showed marked increase in thymine dimer, which decreased by about 50% after 24 h. This decrease in thymine dimer was significantly attenuated ( P < 0.05) by the pretreatment of NH 2Cl (200 μM), which indicated DNA repair inhibition. UVB induced p53 phosphorylation at Ser15, Ser20 and Ser37, and p53 accumulation, and NH 2Cl also inhibited these changes. Consequently, UVB-induced increase in the downstream effectors of p53, namely p21 Cip1 and Gadd45a, were almost completely inhibited by NH 2Cl. Immunoprecipitation study indicated that the association of p53 and MDM2, an E3 ubiquitin ligase for p53, did not change substantially by NH 2Cl and/or UVB. The phosphorylation of p53 (Ser15 and Ser37) by UVB is catalyzed by ATR (ataxia telangiectasia mutated and Rad3 related kinase), which works as DNA damage sensor, and ATR also phosphorylates checkpoint kinase 1(Chk1) at Ser345. NH 2Cl also inhibited the phosphorylation of Chk1 (Ser345). As UVB-induced DNA damage is repaired by nucleotide excision repair (NER) in human cells, these findings indicated that NH 2Cl inhibited NER through the inhibition of p53 phosphorylation and accumulation, and NH 2Cl probably impaired DNA damage recognition and/or ATR activation. NH 2Cl may facilitate carcinogenesis through the inhibition of NER that repairs DNA damages from various carcinogens.

Cancer-Specific Functions of SIRT1 Enable Human Epithelial Cancer Cell Growth and Survival

SIRT1 is a conserved NAD-dependent deacetylase that regulates life span in accord with nutritional provision. In mammalian cells, SIRT1 also down-regulates stress-induced p53 and FoxO pathways for apoptosis, thus favoring survival under stress. The functioning of SIRT1 under normal, nonstressed conditions of cell growth is unknown. Here we have asked if SIRT1 has the capacity to influence cell viability in the absence of applied stress. For this purpose we used synthetic small interfering RNA to silence SIRT1 gene expression by RNA interference (RNAi). We show that the process of RNAi, by itself, does not affect cell growth and is not sufficient to activate a cellular stress response (indicated by lack of activation of endogenous p53). We also show that, in the absence of applied stress, SIRT1 silencing induces growth arrest and/or apoptosis in human epithelial cancer cells. In contrast, normal human epithelial cells and normal human diploid fibroblasts seem to be refractory to SIRT1 silencing. Combined gene knockout with RNAi cosilencing experiments indicate that SIRT1 and Bcl-2 may suppress separable apoptotic pathways in the same cell lineage and that the SIRT1-regulated pathway is independent of p53, Bax, and caspase-2. Alternatively, SIRT1 may suppress apoptosis downstream from these apoptotic factors. In either case, we show that FoxO4 (but not FoxO3) is required as proapoptotic mediator. We further identify caspase-3 and caspase-7 as downstream executioners of SIRT1/FoxO4-regulated apoptosis. Our work identifies SIRT1 as a novel target for selective killing of cancer versus noncancer epithelial cells.

Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts

Using a pulsed electron beam, we investigated the dependence of micronucleus formation on the incident electron energy in AG01522 human diploid fibroblasts after nontargeted irradiations at 25 and 80 keV. Examining the dose response, we found that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the electron track end is responsible for the biological damage occurring in the cell.

Effects of Human Na+/Dicarboxylate Cotransporter 3 on the Replicative Senescence of Human Embryonic Lung Diploid Fibroblasts

To investigate the role of human Na(+)/dicarboxylate cotransporter 3 (hNaDC3) in the replicative senescence of normal human embryonic lung diploid fibroblasts (WI-38), a retroviral vector containing hNaDC3 was constructed. hNaDC3 was introduced into normal WI-38 cells through infection with the retroviral virus. Monoclones were selected with G418. The integration and expression of exotic genes were confirmed by Northern blot and Western blot. When compared with the control cells, WI-38 cells transfected with hNaDC3 cDNA showed significant suppression of growth rate (by 40%), increase of positive rate of SA-beta-gal staining, decrease of mitochondrial membrane potential, shortening of telomere length, and increase of P16 and P21 expression. The morphology characteristics of senescent fibroblasts appeared earlier. Our results have, for the first time, demonstrated that high expression of hNaDC3 may be able to, at least partly, promote the cellular senescence of human diploid fibroblasts.

Functional Analysis of the p53 Pathway in Response to Ionizing Radiation in Uveal Melanoma

Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells. The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-x(L). No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-x(L) in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway. Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.

RNA Silencing of Checkpoint Regulators Sensitizesp53-Defective Prostate Cancer Cells to Chemotherapy while Sparing Normal Cells

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

Induction of prosurvival molecules by apoptotic stimuli: involvement of FOXO3a and ROS

Most cancer therapeutics fails to eradicate cancer because cancer cells rapidly develop resistance to its proapoptotic effects. The underlying mechanisms remain incompletely understood. Here we show that three representative apoptotic stimuli, that is, serum starvation, a mitochondrial toxin, and a DNA-damaging agent (etoposide), rapidly induce several distinct classes of prosurvival molecules, in particular, Bcl-2/Bcl-X(L) and superoxide dismutase (SOD; including both MnSOD and Cu/ZnSOD). At the population level, the induction of these prosurvival molecules occurs prior to or concomitant with the induction of proapoptotic molecules such as Bim and Bak. Blocking the induction using siRNAs of the prosurvival or proapoptotic molecules facilitates or inhibits apoptosis, respectively. One master transcription factor, FOXO3a, is involved in the transcriptional activation of some of these prosurvival (e.g., MnSOD) and proapoptotic (e.g., Bim) molecules. Interestingly, in all three apoptotic systems, FOXO3a itself is also upregulated at the transcriptional level. Mechanistic studies indicate that reactive oxygen species (ROS) are rapidly induced upon apoptotic stimulation and that ROS inhibitors/scavengers block the induction of FOXO3a, MnSOD, and Bim. Finally, we show that apoptotic stimuli also upregulate prosurvival molecules in normal diploid human fibroblasts and at subapoptotic concentrations. Taken together, these results suggest that various apoptotic inducers may rapidly mobilize prosurvival mechanisms through ROS-activated master transcription factors such as FOXO3a. The results imply that effective anticancer therapeutics may need to combine both apoptosis-inducing and survival-suppressing strategies.

Aurora B Overexpression Associates with the Thyroid Carcinoma Undifferentiated Phenotype and Is Required for Thyroid Carcinoma Cell Proliferation

Alterations in chromosome number (aneuploidy) are common in human neoplasias. Loss of mitotic regulation is believed to induce aneuploidy in cancer cells and act as a driving force during the malignant progression. The serine/theronine protein kinases of aurora family genes play a critical role in the regulation of key cell cycle processes. Aurora B mediates chromosome segregation by ensuring orientation of sister chromatids and overexpression of Aurora B in diploid human cells NHDF (normal human diploid fibroblast) induces multinuclearity. We analyzed Aurora B expression in human thyroid carcinomas. Cell lines originating from different histotypes showed an increase in Aurora B expression. Immunohistochemical analysis of archive samples showed a high expression of Aurora B in anaplastic thyroid carcinomas; conversely, Aurora B expression was not detectable in normal thyroid tissue. Real-time PCR analysis confirmed a strong expression of Aurora B in anaplastic thyroid carcinomas. The block of Aurora B expression induced by RNA interference or by using an inhibitor of Aurora kinase activity significantly reduced the growth of thyroid anaplastic carcinoma cells.

Flavin-containing oxidases regulate progression from G 1 to S phase of the cell cycle in normal human diploid fibroblasts

The effects of flavin-containing oxidase enzymes on cellular progression from G 1 to S phase of the cell cycle were examined in normal human diploid fibroblasts by use of the cumulative labeling index method. Significant transient arrests in G 1 occurred when quiescent cell populations incubated with diphenyliodonium (DPI) for a 20 h period at concentrations as low as 0.05 μM were stimulated to proliferate by subculturing to low density in control growth medium. Quiescent cells pre-treated with DPI and subcultured in DPI-containing growth medium were permanently arrested in G 1. Induction of this G 1 checkpoint was associated with activation of the p53/p21 Waf1 signaling pathway. Incubation of G 0/G 1 phase cells with DPI (0.05–0.2 μM) resulted in significant decreases in the expression levels of cyclin D1 and p21 Waf1; no effect was detected on cyclin A expression. Collectively, these results suggest that DPI-sensitive signaling primarily regulates early G 1 events that lead to S-phase transition. Furthermore, they support a critical role for membrane originating oxidants in the regulation of cellular proliferation pathways.

Werner syndrome cells escape hydrogen peroxide-induced cell proliferation arrest.

Werner syndrome (WS) is a rare disease caused by the lack of a functional nuclear WS protein (WRN). WS is characterized by the early onset of premature aging signs and a high incidence of sarcomas. WS diploid fibroblasts have a short life span and extensive genomic instability. Mammalian cells are continuously exposed to reactive oxygen species (ROS), which represent human mutagens and are thought to be a major contributor to the aging process. Hydrogen peroxide (H2O2) is a common ROS intermediate generated by various forms of oxidative stress. In response to H2O2-induced DNA damage, normal human diploid fibroblasts follow a pathway leading to irreversible proliferation arrest and premature senescence. Here we show that in contrast to normal human fibroblasts, WS diploid fibroblasts continue proliferating after extensive H2O2-induced DNA damage and accumulate oxidative DNA lesions. A direct role of WRN in this abnormal cellular response to H2O2 is demonstrated by interfering with WRN expression in normal human fibroblasts. We propose a role for WRN in the detection and/or processing of oxidative DNA lesions and in cellular responses to H2O2 as they relate to some of the phenotypical aspects of WS cells.

Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging.

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.

No increase in senescence-associated beta-galactosidase activity in Werner syndrome fibroblasts after exposure to H2O2.

Normal human diploid fibroblasts (HDFs) exposed to a single H(2)O(2) subcytotoxic stress display features of premature senescence, termed stress-induced premature senescence (SIPS). In this work, our aim was to study SIPS in Werner syndrome (WS) fibroblasts, derived from a patient with WS, a disease resembling accelerated aging. The subcytotoxic dose for WS fibroblasts was found to be inferior to that of normal HDFs, indicating WS fibroblasts are more sensitive to hydrogen peroxide than normal HDFs. SA beta-gal activity has been shown to occur both in vitro and in vivo, and we studied the proportion of WS cells positive for SA beta-gal. Intriguingly, the percentage of positive cells did not increase with the dose of H(2)O(2) used. Contrary to other HDFs, the DNA-binding activity of p53 in WS fibroblasts did not increase in SIPS. We found, based on our results, that WS fibroblasts feature an altered stress response and do not reach SIPS from H(2)O(2). We suggest that the proportion of cells that in normal HDFs would enter SIPS instead die in WS fibroblasts. Last, we propose that aging derives from a loss of integrity of the chromatin structure, which occurs faster in WS patients.

Inhibition of phosphatidylinostol 3-kinase uncouples H 2O 2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H 2O 2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated β-galactosidase (SA-β-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21 Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16 Ink4a, p27 Kip1, and p14 Arf were observed. Exposure of WI38 cells to H 2O 2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H 2O 2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-β-gal activity, but did not affect H 2O 2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H 2O 2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H 2O 2 and does not contribute to the maintenance of permanent cell cycle arrest.

TNF-alpha in vivo stimulates apoptosis in fibroblasts through caspase-8 activation and modulates the expression of pro-apoptotic genes.

Apoptosis of matrix producing cells is common among many inflammatory diseases. The goal of the present study was to examine the apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) on fibroblastic cells in vivo and to investigate the role of different caspases in this process. This was accomplished in vivo by subcutaneous injection of TNF-alpha in mice. The direct effects of TNF-alpha on fibroblast apoptosis were studied in vitro with normal diploid human fibroblasts. The results indicate that TNF-alpha in vivo induces apoptosis of fibroblasts. By RNase protection assay, we demonstrated that TNF-alpha stimulates expression of 12 apoptotic genes. Fluorometric studies demonstrated that TNF-alpha in vivo predominantly increased caspase-8 and -3 activity and by use of specific inhibitors, the activation of caspase-3 was shown to be initiated by caspase-8 with only a minor contribution from caspase-9. Thus, TNF-alpha acts to modulate the expression of many genes that favors apoptosis of fibroblastic cells, which is dependent mostly upon signaling through caspase-8.

Telomere length assessment in tissue sections by quantitative FISH: image analysis algorithms.

Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes. Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm. Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low. Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease.

The genotoxic effect of carcinogenic PAHs, their artificial and environmental mixtures (EOM) on human diploid lung fibroblasts

The aim of this study was to investigate the genotoxic effect of single carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), as well as their binary and artificial mixtures containing c-PAHs in the same relative proportions as real complex mixtures, and to investigate the genotoxic effect of environmental mixtures such as EOM (extractable organic matter adsorbed to respirable air particles). We used normal human diploid lung fibroblasts (HEL) in a confluent (“quiescent”) state as being closer to in vivo conditions. We determined DNA adducts by 32 P -postlabelling and the expression of p53 and p21 WAF1 proteins by Western blotting. Our results showed that the DNA binding potencies of c-PAHs in quiescent HEL cells were as follows: DB[al]P⪢B[a]P⪢B[b]F∼CHRY∼B[k]F∼B[a]A∼DB[ah]A∼I[cd]P>B[ghi]P. Both DB[al]P and B[a]P were chosen to assess the genotoxic effect of other c-PAHs when present in binary mixtures. In all cases, co-treatment resulted in significantly lower DNA adduct levels as compared with adduct levels resulting from single compounds. The highest inhibitory effect was found in the presence of DB[ah]A followed by B[k]F. In both artificial and EOM mixtures a strong inhibitory effect of other compounds was observed. BPDE-adduct levels were up to five-fold lower in an artificial c-PAH mixture and up to 10-fold lower in an EOM sample than expected from applying B[a]P alone at the same concentration. These results suggest a competition for the metabolic activation enzymes, their saturation and/or inactivation by PAH metabolites. We did not observe the decrease of DNA adducts and induced levels of both p53 and p21 WAF1 proteins during the post-treatment period up to 6 days following exposure. We suggest that parent c-PAHs accumulate inside cells and that they are further activated by newly synthesised enzymes during the post-treatment period. Therefore, unaltered c-PAHs may act as a reservoir inside the cells for the long-term production of active metabolites. The total DNA adduct levels induced in HEL cells after exposure to EOM were generally low as compared with exposure to B[a]P alone under the same conditions. Human diploid lung fibroblasts possess low metabolic capacity than other human target cells. Therefore, it may be easier to competitively inhibit the metabolising enzymes in this cell system. We hypothesise that this finding might explain the generally low PAH–DNA adduct levels detected in human studies using surrogate cells such as blood WBC or lymphocytes that also possess low metabolic capacity.

Invasion of normal human fibroblasts induced by v-Fos is independent of proliferation, immortalization, and the tumor suppressors p16INK4a and p53.

Invasion is generally perceived to be a late event during the progression of human cancer, but to date there are no consistent reports of alterations specifically associated with malignant conversion. We provide evidence that the v-Fos oncogene induces changes in gene expression that render noninvasive normal human diploid fibroblasts highly invasive, without inducing changes in growth factor requirements or anchorage dependence for proliferation. Furthermore, v-Fos-stimulated invasion is independent of the pRb/p16(INK4a) and p53 tumor suppressor pathways and telomerase. We have performed microarray analysis using Affymetrix GeneChips, and the gene expression profile of v-Fos transformed cells supports its role in the regulation of invasion, independent from proliferation. We also demonstrate that invasion, but not proliferation, is dependent on the activity of the up-regulated epidermal growth factor receptor. Taken together, these results indicate that AP-1-directed invasion could precede deregulated proliferation during tumorigenesis and that sustained activation of AP-1 could be the epigenetic event required for conversion of a benign tumor into a malignant one, thereby explaining why many malignant human tumors present without an obvious premalignant hyperproliferative dysplastic lesion.