Normal Fibroblast Strains Research Articles

Synthesis, cytotoxic activity evaluation and mechanistic investigation of novel 3,7-diarylsubstituted 6-azaindoles

A number of new disubstituted 6-azaindoles have been designed and synthesized bearing a crucial structural modification in respect to an analogous antiproliferative hit compound. The synthesis was performed using 2-amino-3-nitro-4-picoline, that was suitably modified and converted to 7-chloro-3-iodo-6-azaindole and this central scaffold was used for successive Suzuki-type couplings, to result in the target compounds. The evaluation of the cytotoxic activity was performed against four human cancer cell lines, as well as a normal human fibroblast strain. Certain compounds possessed strong anticancer activity without affecting normal cells. At subcytotoxic concentrations for cancer cells, these compounds displayed an anti-proliferative effect by arresting the cells at the G2/M phase of the cell cycle, which could be associated with the observed decrease in the phosphorylation levels of the MEK1- ERK1/2 pathway and/or the activation of the p53-p21WAF1 axis.

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Simple SummaryA clinical test for alternative-lengthening-of-telomeres (ALT) could assist with cancer diagnosis and monitoring of disease progression. ALT-targeted anticancer treatments are being developed; however, there is no appropriate companion ALT diagnostic. The C-Circle biomarker is the only known ALT specific molecule and the C-Circle Assay the only quantitative ALT assay that is amenable to clinical use. We show here that C-Circles are secreted by ALT+ cancer cell lines inside the exosomes and are protected from nucleases. We also show that secreted C-Circles, like intracellular C-Circles, are an ALT-specific biomarker, and in high-risk neuroblastoma, the blood-based C-Circle Assay has the potential to be an accurate diagnostic for ALT cancer activity. Therefore, the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and, potentially, a clinical diagnostic for ALT activity, which is required for the development of ALT-targeted therapies as well as for the diagnosis and monitoring of ALT+ cancer.C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

The G-quadruplex-stabilising agent RHPS4 induces telomeric dysfunction and enhances radiosensitivity in glioblastoma cells

G-quadruplex (G4) interacting agents are a class of ligands that can bind to and stabilise secondary structures located in genomic G-rich regions such as telomeres. Stabilisation of G4 leads to telomere architecture disruption with a consequent detrimental effect on cell proliferation, which makes these agents good candidates for chemotherapeutic purposes. RHPS4 is one of the most effective and well-studied G4 ligands with a very high specificity for telomeric G4. In this work, we tested the in vitro efficacy of RHPS4 in astrocytoma cell lines, and we evaluated whether RHPS4 can act as a radiosensitising agent by destabilising telomeres. In the first part of the study, the response to RHPS4 was investigated in four human astrocytoma cell lines (U251MG, U87MG, T67 and T70) and in two normal primary fibroblast strains (AG01522 and MRC5). Cell growth reduction, histone H2AX phosphorylation and telomere-induced dysfunctional foci (TIF) formation were markedly higher in astrocytoma cells than in normal fibroblasts, despite the absence of telomere shortening. In the second part of the study, the combined effect of submicromolar concentrations of RHPS4 and X-rays was assessed in the U251MG glioblastoma radioresistant cell line. Long-term growth curves, cell cycle analysis and cell survival experiments, clearly showed the synergistic effect of the combined treatment. Interestingly the effect was greater in cells bearing a higher number of dysfunctional telomeres. DNA double-strand breaks rejoining after irradiation revealed delayed repair kinetics in cells pre-treated with the drug and a synergistic increase in chromosome-type exchanges and telomeric fusions. These findings provide the first evidence that exposure to RHPS4 radiosensitizes astrocytoma cells, suggesting the potential for future therapeutic applications.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.

HPV-positive HNSCC cell lines but not primary human fibroblasts are radiosensitized by the inhibition of Chk1

PurposeDespite the comparably high cure rates observed for HPV-positive HNSCC, there is still a great need for specific tumor radiosensitization due to the often severe side effects resulting from intense radiochemotherapy. We recently demonstrated that HPV-positive HNSCC cell lines are characterized by a defect in DNA double-strand break repair associated with a pronounced G2-arrest. Here we tested whether abrogation of this radiation-induced G2-arrest by the inhibition of Chk1 results in specific radiosensitization of HPV-positive HNSCC cells. Materials and methodsExperiments were performed with five HPV and p16-positive (93-VU-147T, UM-SCC-47, UT-SCC-45, UD-SCC-2, UPCI-SCC-154) and two HPV and p16-negative HNSCC cell lines, as well as two normal human fibroblast strains. Chk1 was inhibited by the selective inhibitor PF-00477736. Cell cycle distribution was determined by flow cytometry, Chk1-activity via Western blot and cell survival by colony formation assay. ResultsWith the exception of UPCI-SCC-154, the inhibition of Chk1 was found to abolish the pronounced radiation-induced G2-arrest in all HPV-positive cells utilized. All tumor cell lines that demonstrated the abrogation of G2-arrest also demonstrated radiosensitization. Notably, in G1-arrest-proficient normal human fibroblasts no radiosensitization was induced. ConclusionAbrogation of the G2 checkpoint through the inhibition of Chk1 may be used to selectively increase the cellular radiosensitivity of HPV-positive HNSCC without affecting the surrounding normal tissue.

Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation

The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ΔΔCq/Normfinder and ΔΔCq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2Gy, and changes in gene expression were analyzed 24h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ΔΔCq/Genorm or ΔΔCq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested.

Ovarian cancer cells, not normal cells, are damaged by Mirk/Dyrk1B kinase inhibition

Prior studies had shown that the Mirk/dyrk1B gene is amplified/upregulated in about 75% of ovarian cancers, that protein levels of this kinase are elevated in quiescent G0 cells and that Mirk maintains tumor cells in quiescence by initiating rapid degradation of cyclin D isoforms and by phosphorylation of a member of the DREAM complex. Depletion of Mirk/dyrk1B led to increased cyclin D levels, an elevated reactive oxygen species (ROS) content and loss of viability. However, many normal cells in vivo are quiescent, and therefore, targeting a kinase found in quiescent cells might be problematic. In our study, Mirk kinase activity was found to be higher in ovarian cancer cells than in normal cells. Pharmacological inhibition of Mirk/dyrk1B kinase increased cyclin D levels both in quiescent normal diploid cells and in quiescent CDKN2A-negative ovarian cancer cells, but led to more active CDK4/cyclin D complexes in quiescent ovarian cancer cells, allowing them to escape G0/G1 quiescence, enter cycle with high ROS levels and undergo apoptosis. The ROS scavenger N-acetyl cysteine reduced both the amount of cleaved poly(ADP-ribose) polymerase (PARP) and the extent of cancer cell loss. In contrast, normal cells were spared because of their expression of cyclin directed kinase (CDK) inhibitors that blocked unregulated cycling. Quiescent early passage normal ovarian epithelial cells and two strains of quiescent normal diploid fibroblasts remained viable after the inhibition of Mirk/dyrk1B kinase, and the few cells that left G0/G1 quiescence were accumulated in G2+M. Thus, inhibition of Mirk kinase targeted quiescent ovarian cancer cells.

No Effect of the Transforming Growth Factor β1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

To study whether the promoter polymorphism (C-509T) affects transforming growth factor β1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

Abstract 2858: Mirk/dyrk1B kinase inhibition increases ROS and apoptosis in ovarian cancer cells, but has little effect on normal diploid cells

Abstract Quiescent cancer cells in vivo, including stem cell populations, are relatively insensitive to most chemotherapeutic drugs and radiation. Tumor cells in culture can be placed in G0 by serum or nutrient deprivation, and then can be targeted by a small molecule Mirk kinase inhibitor. Mirk/dyrk1B levels are elevated up to 10-fold in quiescent tumor cells where Mirk blocks cell cycling and increases viability by decreasing reactive oxygen species (ROS). In prior studies Mirk was detected in 75% of ovarian cancers, and was amplified in a subset. In contrast, Mirk expression was quite low in most normal cell types and Mirk knockout or depletion had no detectable effects on normal cells. Response to a Mirk kinase inhibitor was compared between two strains of normal diploid fibroblasts, early passage cultures of normal human ovarian epithelial cells, and a series of ovarian cancer cell lines where Mirk had been shown to be an active kinase. Serum-starved normal diploid cells entered a G0 quiescent state and had minimal response to the Mirk inhibitor, but serum-starved cancer cells were kept in cycle by the Mirk inhibitor, which reduced E2F4 sequestration by p130/Rb2 by 2-3fold. Mirk inhibitor treated cancer cultures had several-fold more sub-G0/G1 apoptotic cells and about 30-fold higher levels of the apoptotic proteins cleaved PARP and cleaved caspase 3 compared with normal diploid cells. The Mirk inhibitor increased total ROS levels and superoxide levels only in the cancer cells, in a rapid time-course of 2-3 days. Cancer cell loss after Mirk kinase inhibitor treatment was due at least in part to ROS, since the ROS scavenger N-acetyl cysteine reduced the amount of cleaved PARP and the extent of cancer cell loss. The Mirk inhibitor decreased the EC50 of cisplatin up to 9-fold in ovarian cancer cells. Thus ovarian cancer cells, which exhibit elevated Mirk expression/amplification and active Mirk kinase, were shown to be more susceptible to the Mirk kinase inhibitor than normal diploid cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2858. doi:1538-7445.AM2012-2858

Androgen Receptor Mutations Are Associated with Altered Epigenomic Programming as Evidenced by HOXA5 Methylation

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.

Chromosomal instability and telomere lengths of each chromosomal arm measured by Q-FISH in human fibroblast strains prior to replicative senescence

We monitored the telomere lengths and chromosomal instability characteristics of fibroblasts at different population doubling levels (PDLs) to gain further insight into the role of telomere shortening in chromosomal instability. We used 7 normal diploid human fibroblast strains (TIG-1, 3, 7, 103, 104, 112, and 114) and a quantitative fluorescence in situ hybridization method to measure telomere lengths of the p- and q-arms of individual chromosomes. We also enumerated morphologic signs of chromosomal instability, including fusion or loss of chromosomes, and anaphase bridges. In strains TIG-1, 3, 7, 103, and 114 at the late (phase 3) stage (≧40 PDLs), 29 (96.6%) of 30 fusions were associated with one or both of the chromosomal arms that bear significantly shorter telomeres in those populations. In TIG-1 at 62 PDL, 6 fusions were associated with Xq ( n = 3), 21q ( n = 3), and other ( n = 6) chromosomes. Xq and 21q had significantly shorter telomeres, and anaphase bridges were often associated with chromosomes X and/or 21 (74.6%). Our results indicate that chromosomes having excessively shortened telomeres at late PDLs begin to show features of instability such as fusions and anaphase bridges.

Expression of RAD51, BRCA1 and P53 does not correlate with cellular radiosensitivity of normal human fibroblasts

To evaluate the potential role of key DNA repair proteins in the sensitivity of normal human fibroblasts to ionising radiations. Radiosensitivity of six human fibroblast strains established from skin biopsies of women who had undergone conservative breast surgery and received a curative breast conserving radiotherapy was measured by colony-formation assay. The expression level of RAD51, BRCA1 and p53 proteins were studied using western blot analysis. The six fibroblast strains represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy (SF3.5) ranging from 0.21 to 0.40. We found that these differences in cell survival did not correlate with the expression of RAD51, BRCA1 nor p53 in the tested normal human fibroblast strains. We conclude that measurement of protein expression of the three tested genes (RAD51, BRCA1 and p53) did not reflect sensitivity of normal fibroblasts to IR.

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results.

Abstract B190: Efficacy of the specific ETA receptor antagonist zibotentan (ZD4054) in cancer cells and fibroblasts from colorectal cancer

Abstract Endothelin-1 (ET-1) contributes to growth and progression of solid cancers, mainly through endothelin receptor A (ETAR). Therefore endothelin receptor antagonism is emerging as a potential treatment for neoplasms. We evaluated the efficacy of the specific ETAR antagonist zibotentan (ZD4054) in blocking ET-driven cellular effects in colorectal cancer (CRC). CRC cell lines (HT29, SW620) and primary normal fibroblast strains isolated from human colorectal tissues (CF36, CF56, CF65, CF75) were incubated in ET-1 with/without BQ123, zibotentan (ETAR antagonists), BQ788 (ETBR antagonist). Resultant cell growth was measured by the colourimetric methylene blue assay; migration by a modified monolayer scratch assay; contraction in collagen gels; downstream effectors by western blotting. ET-1 driven growth (18%–45% above control) was significantly inhibited (p<0.01) by ETAR (not ETBR) antagonism (BQ123=zibotentan CRC; fibroblasts). ET-1 driven fibroblast migration and contraction were blocked by ETAR and ETBR antagonism (zibotentan=BQ123). CRC cells did not migrate or contract. ET-1-stimulated expression of downstream effectors was driven by ETAR or ETBR, eg: (1) connective tissue growth factor (CTGF) was blocked by ETAR antagonism (zibotentan>BQ123; CRC and fibroblasts); (2) collagen XI was blocked by ETAR>ETBR antagonism (zibotentan>BQ123; fibroblasts). The specific ETAR antagonist zibotentan is at least as efficacious as BQ123 in blocking ET-1 driven growth, migration and contraction both in CRC cells and colorectal fibroblasts, which form the supporting tumor stroma. Zibotentan is a strong candidate for adjuvant treatment in CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B190.

Differences in the Nemosis Response of Normal and Cancer-Associated Fibroblasts from Patients with Oral Squamous Cell Carcinoma

BackgroundTumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.Principal FindingsFibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.ConclusionsNemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers α-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.

Apolipoprotein D (APOD) is a putative biomarker of androgen receptor function in androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is the most common cause of disorders of sex development usually caused by mutations in the androgen receptor (AR) gene. AIS is characterized by a poor genotype-phenotype correlation, and many patients with clinically presumed AIS do not seem to have mutations in the AR gene. We therefore aimed at identifying a biomarker enabling the assessment of the cellular function of the AR as a transcriptional activator. In the first step, we used complementary DNA (cDNA) microarrays for a genome-wide screen for androgen-regulated genes in two normal male primary scrotal skin fibroblast strains compared to two labia majora fibroblast strains from 46,XY females with complete AIS (CAIS). Apolipoprotein D (APOD) and two further transcripts were significantly upregulated by dihydrotestosterone (DHT) in scrotum fibroblasts, while CAIS labia majora cells were unresponsive. Microarray data were well correlated with quantitative real-time polymerase chain reaction (qRT-PCR; R = 0.93). Subsequently, we used qRT-PCR in independent new cell cultures and confirmed the significant DHT-dependent upregulation of APOD in five normal scrotum strains [13.5 +/- 8.2 (SD)-fold] compared with three CAIS strains (1.2 +/- 0.7-fold, p = 0.028; t test) and six partial androgen insensitivity syndrome strains (2 +/- 1.3-fold, p = 0.034; t test). Moreover, two different 17ss-hydroxysteroid dehydrogenase III deficiency labia majora strains showed APOD induction in the range of normal scrotum (9.96 +/- 1.4-fold), supporting AR specificity. Therefore, qRT-PCR of APOD messenger RNA transcription in primary cultures of labioscrotal skin fibroblasts is a promising tool for assessing AR function, potentially allowing a function-based diagnostic evaluation of AIS in the future.

The anti-apoptotic role for p53 following exposure to ultraviolet light does not involve DDB2

The p53 tumour suppressor is a transcription factor that can either activate or repress the expression of specific genes in response to cellular stresses such as exposure to ultraviolet light. The p53 protein can exert both pro- and anti-apoptotic effects depending on cellular context. In primary human fibroblasts, p53 protects cells from UV-induced apoptosis at moderate doses but this is greatly affected by the nucleotide excision repair (NER) capacity of the cells. The damage-specific DNA binding protein 2 (DDB2) is involved in NER and is associated with xeroderma pigmentosum subgroup E (XP-E). Importantly, DDB2 is also positively regulated by the p53 protein. To study the potential interplay between DDB2 and p53 in determining the apoptotic response of primary fibroblasts exposed to UV light, the expression of these proteins was manipulated in primary normal and XP-E fibroblast strains using human papillomavirus E6 protein (HPV-E6), RNA interference and recombinant adenoviruses expressing either p53 or DDB2. Normal and XP-E fibroblast strains were equally sensitive to UV-induced apoptosis over a broad range of doses and disruption of p53 in these strains using HPV-E6 or RNA interference led to a similar increase in apoptosis following exposure to UV light. In contrast, forced expression of p53 or DDB2 did not affect UV-induced apoptosis greatly in these normal or XP-E fibroblast strains. Collectively, these results indicate that p53 is primarily protective against UV-induced apoptosis in primary human fibroblasts and this activity of p53 does not require DDB2.

UV-inducible base excision repair of oxidative damaged DNA in human cells

Methylene blue (MB) acts as a photosensitizer and after excitation by visible light (VL) produces reactive oxygen species that result in oxidatively damaged DNA. (MB + VL) produces predominantly 8-hydroxyguanine as well as other single base modifications in DNA that are repaired by base excision repair (BER). We have used a recombinant non-replicating human adenovirus, Ad5HCMVlacZ, which expresses the beta-galactosidase (beta-gal) reporter gene, to examine the role of the p53 tumor suppressor in constitutive and inducible BER of MB + VL-damaged DNA in human cells. Host cell reactivation (HCR) of beta-gal activity for MB + VL-treated Ad5HCMVlacZ was examined in normal human fibroblasts and several transformed and tumor cell lines with compromised p53 function using both non-treated cells and cells pretreated with ultraviolet light of 200-280 nm wavelength (UVC). Constitutive HCR of the MB + VL-treated reporter gene in untreated cells did not correlate with wild-type p53 expression levels, suggesting that factors other than p53 expression levels can influence constitutive BER of the reporter gene. UVC pre-treatment of the normal fibroblast strains resulted in an enhanced HCR of the MB + VL-treated reporter gene and a concomitant increase in the expression of p53, suggesting that p53 may be involved in UV-inducible BER in normal human fibroblasts. In contrast, p53 expression did not correlate with HCR values for the p53-compromised cells in UVC-pre-treated cells. In particular, the SKOV-3, LFS 087 and NF-E6 cells showed no up-regulation of p53 expression following UVC, and yet these cells showed significant enhancement of HCR following UVC pre-treatment. These results indicate that BER of MB + VL-damaged DNA is inducible in human cells by pre-UVC treatment and that the enhancement in BER may result from both p53-dependent and p53-independent mechanisms.

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

Background and purpose The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins. Materials and methods Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. Results The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15 Gy) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor in the activity of the DNA-PK complex induced upon irradiation. Conclusions For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity.