Cultures Of Normal Human Fibroblasts Research Articles

EFFICIENCY OF PHOTON CAPTURE BEAM TECHNOLOGY AND PHOTODYNAMIC IMPACT ON MALIGNANT AND NORMAL HUMAN CELLS IN VITRO.

to investigate the structural and morphofunctional changes in test systems of malignant (cell lineA549) and normal (stem fibroblasts) human cells exposed to X-rays in the presence of gadoliniumcontaining photon capture agent «Dotavist» and optical light (red spectrum) in combination with «Fotolon» photosensitizer. The continuous cell culture of normal human fibroblasts and malignant human cells technology, X-ray and red light exposure, cytological and statistical methods. Effects of the two binary radiation technologies, namely the photon capture impact on malignant cells(human nonsmall cell lung cancer cells i.e. line A-549) and normal cells (human stem fibroblasts) when incubated with gadoliniumcontaining photon capture agent «Dotavist» and photodynamic effect in the presence of «Fotolon» photosensitizer applied separately and in combination were studied in a comparative mode. Proceeding from morphofunctional characteristics (growth kinetics, proliferative and mitotic activity) of the abovementioned test systems, peculiarities of the effect on malignant and normal cells were established. Irradiation with X-rays to the 1.0, 5.0, and 10.0 Gy doses resulted in inactivation of respectively 10 %, 46 %, and 80% of the A-549 line malignant cells.Cellular irradiation to a 1.0 Gy dose in the presence of the photon capture agent «Dotavist» (10 μl/ml concentration) inhibited cell proliferation by 50 %, suppressing their mitotic activity. At a dose of 10.0 Gy in the presence of «Dotavist» the inhibition by 93 % of the growth and division of malignant cells occurred, indicating the high efficiency of binary radiation technology. The effect of two binary radiation technologies on malignant human cells (A-549 line), namely the combination of red light with «Fotolon» (0.05 mg/ml concentration) and X-ray exposure in the above doses with «Dotavist» (10 μl/ ml concentration) resulted in the death of respectively 64 %, 86 %, and 99% malignant cells. The culture of normal fibroblasts was found being more sensitive to the influence of a complex of binary radiation impact, as exposure to a dose of 10.0 Gy in the presence of «Dotavist» and «Fotolon» inactivated 100 % of cells. The obtained results provide basis of preclinical evaluation of effectiveness of the combined impact of two binary technologies and drugs used in the photon capture technology and photodynamic therapy i.e. the photon capture agent «Dotavist» and «Fotolon» photosensitizer respectively.

Effect of adalimumab on the expression profile of mRNA, and protein associated with JAK/STAT signaling pathway in fibroblast exposed to lipopolysaccharide.

The aim of this study was to assess the effect of adalimumab on the expression level of mRNA and protein TNF-α, IFN-γ, IL-17, IL12A, IL12B, and IL23A in the culture of normal human fibroblasts, in which the LPS inflammation process was induced. The NHDF culture was exposed to the effect of LPS in the concentrations of 1, 2, and 10 μg/mL for 2, 8, and 24 hour periods, and then adalimumab was added at the concentration of 8 μg/mL, it was then incubated for 2, 8, and 24 hour. Cells unexposed to LPS and adalimumab constituted the control. The microarray expression techniques, RTqPCR, and ELISA assay were used. Irrespectively of the concentration of LPS used and the incubation time of it with cells overexpression of the analyzed genes is present, with increasing factor concentration used to induce inflammation and incubation time with it, expression of the assessed genes was greater. In turn, adding the anti-TNF drug to the culture caused the silencing of the expression of the mRNAs and proteins. It was confirmed that LPS and adalimumab above all affect the expression of genes and proteins dependent on the interaction of IL-12 with receptors, which are TNF-α and IFN-γ, and to a lesser extent also modulate IL-17.

61 - Mitochondrial dynamics regulates metabolism during aging

Cellular metabolism is intimately linked to mitochondrial functions. Mitochondrial dynamics is regulated by mitochondrial fission and fusion that influence the shape, size, and number of mitochondria. Dynamin-related guanosine triphosphatases regulate fission and fusion events: dynamin-related protein 1 (DRP1) regulates fission; mitofusin-1 and 2 (MFN1, MFN2) anchored in the outer mitochondrial membrane and optic atrophy 1 (OPA1) anchored in the inner mitochondrial membrane regulate fusion. Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan (CLS) is defined as the duration during which quiescent normal cells retain their capacity to re-enter the proliferative cycle and then exit back to quiescence. This study investigates whether mitochondrial dynamics regulates metabolism during CLS . Results from bioenergetics measurements of quiescent primary cultures of normal human fibroblasts (NHFs) from subjects of different ages showed significant decreases in the glycolytic flux: 2-10 npH cell-1 s-1 in older (58-y, 61-y, 63-y, 68-y, and 70-y) compared to 12-16 npH cell-1 s-1 in younger (newborn, 3-d, 5-m, and 1-y) subjects. Surprisingly, mitochondrial respiration (oxygen consumption rate; OCR) increased significantly during CLS: 36-77 amol O2 cell-1 s-1 in older compared to 24-29 amol O2 cell-1 s-1 in younger subjects. The decrease in glycolytic flux and increase in OCR during CLS were also associated with a decrease in lactate and increase in ATP levels. Mitochondrial dynamics shifted more towards fusion during CLS. Genetic knock-down of MFN1 and OPA1 in old NHFs decreased OCR and enhanced glycolysis. These results support the hypothesis that mitochondrial dynamics regulates metabolic plasticity during aging; MFN1 and OPA1 regulate this process.

Narciclasine effects on myofibroblast clustering (734.8)

Myofibroblasts are wound‐healing cells that differentiate from fibroblasts. They are characterized by the expression of alpha‐smooth muscle actin stress fibers. Narciclasine is a plant growth modulator that was shown to promote stress fibers in cancer cells and growth arrest in normal cells through the Rho‐kinase pathway. These same characteristics are involved in myofibroblast differentiation. Our goal was to determine whether narciclasine treatment was sufficient to induce myofibroblasts. Adult human fibroblasts were grown on coverslips and treated +/‐ TGF‐beta1 for 24 hours, followed by narciclasine treatment. Myofibroblast differentiation increased from control with narciclasine treatment but no difference was seen when narciclasine was added to TGF‐beta1 treated normal human fibroblast cultures; however, cell clustering was observed in Dupuytren’s treated fibroblasts under these conditions. Work is currently underway to understand the basis for cell clustering in myofibroblasts mediated by narciclasine.

Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures: the impact of radiation quality.

Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036-0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from monochromatic 5.35 keV X ray (LET ~6 keV/μm), 18.3 MeV/u carbon ion (LET ~103 keV/μm), 13 MeV/u neon ion (LET ~380 keV/μm) or 11.5 MeV/u argon ion (LET ~1,260 keV/μm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects.

Nontargeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations

The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/μm] or 600 MeV/u silicon ions (LET ∼50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21(Waf1) (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/μm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/μm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles.

Cadaverine effect on microtubule polymerization

The effect of in vitro age and donor age on net release of adenosine and inosine was studied in cultures of normal human fibroblasts. Confluent cultures of low- (population doubling level [PDL] 23–25) and high- (PDL 43–45) passage human lung fibroblasts derived from a 16-week-old fetal donor (IMR-90) were incubated for 30 min in physiological saline and the release of adenosine and inosine into the saline was determined by HPLC. Release of adenosine and inosine into the saline bathing low-passage human skin fibroblasts derived from a 16-week-old fetal donor (GM6111) was also determined and compared with two strains of low-passage skin fibroblasts from aged (66–67 years) donors (GM3529 and GM3524). The release of adenosine and inosine by low-passage cultures of fetal lung fibroblasts was 911 and 225 pmol/30 min per mg protein, respectively. In high-passage cultures of lung fibroblasts, release of adenosine and inosine was significantly greater at 1403 and 351 pmol/30 min per mg protein, respectively. The release of adenosine and inosine by low-passage cultures of fetal skin fibroblasts was 250 and 179 pmol/30 min per mg protein, respectively. In low-passage skin fibroblasts from aged donors, release of adenosine and inosine was significantly greater at 583 and 652 pmol/30 min per mg protein, respectively. These results indicate that the net release of adenosine and inosine by cultured human fibroblasts into their extracellular environment is enhanced by in vitro aging of lung fibroblasts and is greater in skin fibroblasts from aged donors.

Telomere alterations and genomic instability in long-term cultures of normal human fibroblasts irradiated with X rays and protons

Telomeres are the end of linear chromosomes, responsible for chromosome stability and cell viability. It is well known that radiations are able to induce chromosome instability but it has not yet been investigated whether telomere structure is affected by the radiation exposure and if radiations with different quality act in a different way on telomeres. The effect of radiations with different quality on telomere structure and chromosome instability was analysed in human primary fibroblasts exposed to X rays or low-energy protons (28.5 keV μm(-1)). Telomere length was evaluated at different harvesting times from 24 h up to 360 h (15 days), whereas chromosome instability was evaluated in terms of sister chromatid exchanges (SCEs) (48 h from irradiation) and chromosome painting (360 h from irradiation). Results indicated a delayed telomere lengthening 360 h after X-ray treatment, whereas protons were able to induce such a lengthening shortly from irradiation as well as at longer harvesting times. Data obtained from chromosome instability analysis indicated an increase of SCE frequency only after proton irradiation, but, on the contrary, at the longer harvesting time chromosome painting analysis displayed a higher frequency of aberrations after X-ray treatment, suggesting a role of selective process against highly damaged cells.

Polymeric Drugs Based on Random Copolymers with Antimitotic Activity

Polymeric drugs based on random copolymers with antimitotic activity were obtained by free radical copolymerization of oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside methacrylate (OAGMA) and 2-ethyl-(2-pyrrolidone) methacrylate (EPM) at low and high conversion and analyzed in terms of microstructure, physicochemical, and biological properties. Reactivity ratios of monomers were found to be r(OAGMA) = 1.34 and r(EPM) = 0.98, indicating the obtaining of statistical copolymers with random sequence distribution of the comonomeric units in the macromolecular chains. The glass transition temperature of the copolymers presents a negative deviation from the predicted values according to the Fox equation, suggesting a higher flexibility of the alternating diad. Copolymeric systems with OAGMA contents between 10-50 mol % presented thermosensitive behavior in a heating process showing cloud point temperatures (CPT) in the range 45-28 °C with increasing OAGMA content and hysteresis in one heating-and-cooling cycle. In vitro glycolipid release studies revealed the stability of the ester group in culture medium. The polymeric drugs with 30 and 50 mol % OAGMA presented antimitotic activity on a human glioblastoma line, but they were less toxic on normal human fibroblast cultures.

Propagation Distance of the α-Particle-Induced Bystander Effect: The Role of Nuclear Traversal and Gap Junction Communication

When cell populations are exposed to low-dose alpha-particle radiation, a significant fraction of the cells will not be traversed by a radiation track. However, stressful effects occur in both irradiated and bystander cells in the population. Characterizing these effects, and investigating their underlying mechanism(s), is critical to understanding human health risks associated with exposure to alpha particles. To this end, confluent normal human fibroblast cultures were grown on polyethylene terephthalate foil grafted to an ultrathin solid-state nuclear track detector and exposed under non-perturbing conditions to low-fluence alpha particles from a broadbeam irradiator. Irradiated and affected bystander cells were localized with micrometer precision. The stress-responsive protein p21(Waf1) (also known as CDKN1A) was induced in bystander cells within a 100-microm radius from an irradiated cell. The mean propagation distance ranged from 20 to 40 microm around the intranuclear alpha-particle impact point, which corresponds to a set of approximately 30 cells. Nuclear traversal, induced DNA damage, and gap junction communication were critical contributors to propagation of this stressful effect. The strategy described here may be ideal to investigate the size of radiation-affected target and the relative contribution of different cellular organelles to bystander effects induced by energetic particles, which is relevant to radioprotection and cancer radiotherapy.

Peroxisome proliferator‐activated receptor‐γ abrogates Smad‐dependent collagen stimulation by targeting the p300 transcriptional coactivator

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

A LET-Dependent Decrease in the Apoptotic Response of Normal Human Fibroblast Cultures to Isosurvival Doses of γ-Rays and Energetic Heavy Ions

Biological effectiveness varies with the linear energy transfer (LET) of ionizing radiation. Plentiful evidence has been presented demonstrating that at physically equivalent doses, high-LET energetic heavy ions are more cytotoxic and genotoxic than low-LET photons like X-rays and γ-rays. Notwithstanding, its potential impact at isosurvival doses is yet to be characterized. Here we investigated the cell-killing effectiveness of γ-rays (0.2 keV/μm) and five different beams of heavy ions with LET ranging from 16.2 to 1610 keV/μm in confluent cultures of normal human fibroblasts. The relative biological effectiveness based on the dose giving 10% clonogenic survival peaked at 108 keV/μm. In cultures exposed to the 10% survival doses, the yield of apoptotic cells escalated with time postirradiation but declined with LET. Our results imply that the cell death mode differs with LET at isosurvival levels.

Induction of Extracellular Matrix Synthesis in Normal Human Fibroblasts by Anthraquinone Isolated from Morinda citrifolia (Noni) Fruit

In previous studies we found that Morinda citrifolia (Noni) fruit extract up-regulated biosynthesis of type I collagen and glycosaminoglycans in primary cultures of normal human fibroblasts. The objective of this study was to identify the active ingredients in Noni fruit extract. An active single compound having a type I collagen-stimulating effect was isolated and identified as 1,4-dihydroxy-2-methoxy-7-methylanthraquinone by nuclear magnetic resonance, infrared, and mass analysis. It was revealed that anthraquinone showed significantly increased elaboration of procollagen type I C-terminal peptide and glycosaminoglycans and reduced expression of the collagenase matrix metalloproteinase-1 dose-dependently in human dermal fibroblasts. Furthermore, in a clinical trial, a nano-emulsion containing anthraquinone predominantly increased the dermal type I procollagen in nude mouse skin. These results suggest that anthraquinone derived from Noni extract is a good candidate for use as a new anti-wrinkle agent due to its strong induction of biosynthetic activity of extracellular matrix components.

Autoantibodies to Fibrillin-1 Activate Normal Human Fibroblasts in Culture through the TGF-β Pathway to Recapitulate the “Scleroderma Phenotype”

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.

Gene Targeting by Adeno-Associated Virus Vectors Is Cell-Cycle Dependent

Adeno-associated virus (AAV) vectors can be used to introduce site-specific mutations into homologous chromosomal sequences. There are many potential applications of this technique, but the process of AAV-mediated gene targeting and factors that influence targeting efficiency are not completely understood. We investigated the dependence of AAV-mediated gene targeting on the host cell-cycle status. The frequency of gene targeting by AAV vectors was compared in dividing and serum-arrested normal human fibroblast cultures. Gene targeting occurred in arrested fibroblast cultures at 0.15 to 1.1% the frequency of dividing cultures, and only took place in cells that had undergone DNA synthesis. Gene targeting was also reduced when DNA synthesis was inhibited by hydroxyurea.

The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage

Rabdosia rubescens is a herbal medicine used to treat esophageal cancer in China. In this study, the sesquiterpene oridonin, an isoprenoid, was isolated from Rabdosia rubescens. Mass spectroscopy and carbon 13 NMR spectroscopy were used to identify the structure of the purified compound. It was then evaluated for biological activity against human cell lines derived from prostate (DU-145, LNCaP), breast (MCF-7), and ovarian (A2780 and PTX10) cancers. Oridonin exhibited anti-proliferative activity toward all cancer cell lines tested, with an IC50 estimated by the MTT cell viability assay ranging from 5.8+/-2.3 to 11.72+/-4.8 microM. Flow cytometric analysis demonstrated that oridonin induced a G1 phase arrest in androgen receptor-positive LNCaP cells containing wt p53, while it blocked the cell cycle at G2 and M phases in androgen receptor-negative DU-145 cells with mutated p53; the arrest in M was verified by examination of cell morphology and by the increased frequency of cells with Ser-10 phosphorylated histone H3. The increased incidence of apoptosis, identified by characteristic changes in cell morphology, was seen in tumor lines treated with oridonin. Notably, at concentrations that induced apoptosis among tumor cells, oridonin failed to induce apoptosis in cultures of normal human fibroblasts. Western blot analysis was used to determine the protein expression of cancer suppressor genes, p53 (wt) and Bax, and the proto-oncogene, Bcl-2 in LNCaP cells following treatment with oridonin. Oridonin up-regulated p53 and Bax and down-regulated Bcl-2 expression in a dose-dependent manner. To further explore the possible interaction between oridonin and DNA, its absorption spectrum was measured in the presence and absence of double stranded (ds) DNA. Spectral shifts and an increase in absorption band intensity were observed indicating interaction of oridonin with DNA bases. The nature of the binding is not clear at present though no evidence of histone H2AX phosphorylation on Ser-139 was apparent in DU-145 cells treated with oridonin that would indicate the induction of ds DNA breaks. In conclusion, oridonin inhibits cancer cell growth in a cell cycle specific manner and shifts the balance between pro- and anti-apoptotic proteins in favor of apoptosis. The present data suggest that further studies are warranted to assess the potential of oridonin in cancer prevention and/or treatment.

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G 1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G 1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G 1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0–30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the γH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.

Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis.

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.

Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan.

By immunoscreening with an antibody raised against a plasma membrane protein, we have cloned a growth suppressor gene, Gros1 and assigned it to short arm of human chromosome 1. Two alternatively spliced forms of the gene encoding 84- and 41-kDa (carboxy-terminus truncated) proteins were cloned. The two transcripts, 4.4 and 2.7 kb, were expressed weakly in most of the human tissues, with a high expression of the smaller transcript in placenta, ovary and testis. Normal human fibroblasts in culture showed two transcripts, with a higher level of expression of the 4.4 kb transcript. Transformed cells on the other hand showed predominant expression of the 2.7 kb transcript. Two Gros1 transcripts were also detected in most of the mouse tissues. Stable transfection of the mouse cDNA encoding the 85-kDa protein into NIH3T3 cells resulted in their slow growth and reduced colony-forming efficiency. Stable clones expressing antisense RNA on the other hand exhibited higher colony forming efficiency. While our data implied that Gros1 is a novel growth suppressor gene on human chromosome 1, an independent study has recently characterized its rat-homolog as a leucine proline-enriched novel basement membrane-associated proteoglycan leprecan. We describe here cloning, expression and biological activity analysis implying that this novel proteoglycan is a potential growth suppressor on chromosome 1p31, frequently altered in many malignancies.

Characterization of a polyclonal antibody to human thymidylate synthase suitable for the study of colorectal cancer specimens.

Measurement of thymidylate synthase (hTS) using immunohistochemical techniques has been reported in several clinical studies. However, its value as a prognostic indicator is still not clear. To pursue this, we have developed a new rabbit polyclonal antibody, hTS7.4. The antigen was recombinant hTS containing an N-terminal His(6)-tag. Antiserum hTS7.4 detected recombinant hTS by ELISA at a titer of 1:100,000. Western blot analysis of several human cell lines showed a single band of the expected 36-kD molecular size. HeLa cells treated with the TS inhibitor 5-FUdR showed the expected additional band corresponding to the ternary complex of hTS-dFUMP-reduced folate. hTS7.4 detected TS in bacterial, rat, mouse, and monkey cell extracts, and hTS8.3 (a closely related antiserum) immunoprecipitated a 36-kD [(35)S]-methionine-labeled protein from HeLa extracts. TS was detectable by indirect immunofluorescence in HeLa cells. Proliferating normal human fibroblasts in culture showed staining, but nonproliferating cells did not. Lymphocytes in the germinal center of human tonsil tissue, which are known to be proliferating, stained with hTS7.4 and also with monoclonal antibody TS106. TS may therefore be useful as an immunohistochemical marker of cell proliferation. Normal colon mucosa showed weak staining, whereas some colorectal cancer specimens stained very strongly with hTS7.4. A clinical study of colorectal cancer using this antibody is in progress. (J Histochem Cytochem 47:1563-1573, 1999)