Effects In Normal Human Fibroblasts Research Articles

Biological effects in normal human fibroblasts following chronic and acute irradiation with both low- and high-LET radiation

IntroductionRadiobiological studies at low dose rates allow us to improve our knowledge of the mechanisms by which radiation exerts its effects on biological systems following chronic exposures. Moreover, these studies can complement available epidemiological data on the biological effects of low doses and dose rates of ionizing radiation. Very few studies have simultaneously compared the biological effects of low- and high-LET radiations at the same dose rate for chronic irradiation.MethodsWe compared, for the first time in the same experiment, the effects of chronic (dose rates as low as ~18 and 5 mGy/h) and acute irradiations on clonogenicity and micronucleus formation in AG1522 normal human skin fibroblasts in the confluent state exposed to doses of low- and high-LET radiation (gamma rays and alpha particles) to investigate any differences due to the different radiation quality and different dose rate (in the dose range 0.006–0.9 Gy for alpha particles and 0.4–2.3 Gy for gamma rays).ResultsAs expected, alpha particles were more effective than gamma rays at inducing cytogenetic damage and reduced clonogenic cell survival. For gamma rays, the cytogenetic damage and the reduction of clonogenic cell survival were greater when the dose was delivered acutely instead of chronically. Instead, for the alpha particles, at the same dose, we found equal cytogenetic damage and reduction of clonogenic cell survival for both chronic and acute exposure (except for the highest doses of 0.4 and 0.9 Gy, where cytogenetic damage is greater at a low dose rate).ConclusionThe results of this study may have an impact on space and terrestrial radioprotection of humans at low doses and low dose rates, on biodosimetry, and on the use of ionizing radiation in medicine. These results also provide insights into understanding damage induction and cell reaction mechanisms following chronic exposure (at dose rates as low as 18 and 5 mGy/h) to low- and high-LET radiation.

Lethal and mutagenic bystander effects in human fibroblast cell cultures subjected to low-energy-carbon ions

Purpose: We studied lethal and mutagenic bystander effects in normal human fibroblasts irradiated with low-energy-carbon ions.Materials and methods: After cells reached confluence, cells were irradiated with initial energies of 6 MeV/n carbon ions. The residual energy and LET value were 4.6 MeV/n and 309 keV/µm. The doses used for survival and mutational studies were 0.082 and 0.16 Gy. Irradiation was carried out using 4 different irradiation conditions and plating conditions: (1) The entire cell area on the Mylar film was irradiated (We abbreviate as ‘all irradiation’); (2) Irradiated and unirradiated cells were pooled in a 1:1 ratio and plated as a single culture until the plating for lethal and mutagenic experiments (We abbreviate as ‘mixed population’); (3) Only half of the area on the Mylar film were irradiated using an ion-beam stopper (We abbreviate as ‘half irradiation’); and (4) Only half of the area of the cells were irradiated, and a specific inhibitor of gap junctions was added to the culture (We abbreviate as ‘half irradiation with inhibitor’). Cell samples were analyzed for lethal and mutagenic bystander effects, including a PCR evaluation of the mutation spectrum.Results: The surviving fraction of all irradiation was the same as the half irradiation case. The surviving fractions of both mixed population and the half irradiation with inhibitor were the same level and higher than those of all irradiation and half irradiation. The mutation frequencies at the HPRT (the hypoxanthine-guanine phosphoribosyl transferase) locus of all irradiation and half irradiation were at the same level and were higher than those of mixed population and half irradiation with inhibitor, respectively.Conclusion: There is evidence that the bystander effects for both lethality and mutagenicity occurred in the unirradiated half of the cells, in which only half of the cells were irradiated with the carbon ions. These results suggest that the bystander cellular effects via gap-junction-mediated cell-cell communication are induced by high-LET-carbon ions.

The novel brassinosteroid analog BR4848 inhibits angiogenesis in human endothelial cells and induces apoptosis in human cancer cells in vitro

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17β-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G2/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G2/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERβ, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients.

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1-2Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.

Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis.

Cyclic Pifithrin-α Sensitizes Wild Type p53 Tumor Cells to Antimicrotubule Agent–Induced Apoptosis

As a consequence of multiple functions of p53, its activation in response to cytotoxic stress may have proapoptotic or protective effects depending on the nature of lesions. We have previously shown that mutational inactivation of p53 results in sensitization to paclitaxel. In this study, we used cyclic pifithrin-α, a transcriptional inhibitor of p53, to further investigate the relevance of p53 function in the response of tumor cells to microtubule inhibitors. Using drug concentrations causing only antiproliferative effects, the combination of antimicrotubule agents with subtoxic pifithrin-α doses resulted in increase of sensitivity of two wild type p53 cell lines, associated with a substantial M phase cell accumulation and marked sensitization to apoptosis. Pifithrin-α had no sensitizing effect in p53 defective cells or a marginal effect in normal human fibroblasts. The apoptotic response to the combination was concomitant with p21 down-regulation, Polo-like kinase 1 up-regulation, p34cdc2 kinase dephosphory-lation, and cdc25C phosphatase phosphorylation, supporting mitotic arrest. Sensitization to paclitaxel-induced apoptosis was also achieved by p53-siRNA transfection in wild type p53 H460 cells. Pifithrin-α did not enhance the apoptotic response after p53 down-regulation. The results support a protective role of the transcriptional activity of p53 in response to mitotic spindle damage. The inhibition of transcriptional activity of p53 may have therapeutic implications in the treatment of p53 wild type tumors with antimitotic agents.

Bystander cellular effects in normal human fibroblasts irradiated with low-density carbon ions

We have studied bystander lethal and mutagenic effects in normal human fibroblasts irradiated with low-density carbon ions following four different methods: (1) irradiate with all cells; (2) irradiate with half of cells; (3) mix with two cell populations (1) and (2); (4) treat with the inhibitor of cell–cell communication to half-cell irradiated group. The results of both cell killing and mutation induction showed that there observed in the same effects between (1) and (2). Furthermore, both effects for (4) showed the same level with (3). There is evidence that bystander effects of cell killing and mutation induction occurred in unirradiated half-cells of the irradiation method (2), and cell–cell communication may play an important role of inducing the bystander effects.

Relationship between the radiosensitizing effect of wortmannin, DNA double-strand break rejoining, and p21WAF1 induction in human normal and tumor-derived cells.

Wortmannin (WM) is a potent inhibitor of the catalytic sub-unit of DNA-PK, which is involved in one pathway of DNA double-strand break (DSB) rejoining, and of ATM, which functions upstream in the p53 signaling pathway. WM is known to be an efficient radiosensitizer in a variety of mammalian cell types, to inhibit DSB rejoining following exposure to supralethal doses (> or =30 Gy) of ionizing radiation, and to abrogate the induction of p53 at early times after radiation exposure. We report here that WM is a more effective radiosensitizer in A549 human lung carcinoma cells than in normal human fibroblasts (NHFs). In addition, WM strongly inhibits DSB rejoining in A549 cells exposed to relatively low doses (e.g., 10 Gy) of ionizing radiation, without having any detectable effect in NHFs. We further demonstrate that WM significantly potentiates the induction of p21WAF1, a p53-regulated gene that encodes for a key mediator of cell-cycle/growth arrest, when determined at late times (e.g., 24 h) after irradiation. This late WM-dependent potentiation of p21WAF1 induction following radiation exposure is observed in NHFs and in the p53 wild-type tumor cell lines A549, A172, and SKNSH, but not in the p53-deficient tumor cell lines DLD-1, HeLa, and SKNSH-E6. We conclude that: (i) inhibition of DSB rejoining by WM may be an important contributor to its radiosensitizing effect in A549 cells but not in NHFs; and (ii) radiosensitization of p53-proficient human cells by WM may in part be associated with the delayed induction of p21WAF1, which can lead to a sustained growth-arrested phenotype resembling senescence.

Cellular response in normal human cells exposed to chronically low-dose radiation in heavy-ion radiation field

We have been studying the biological effects in normal human fibroblasts exposed to chronically low-dose radiation in a heavy-ion radiation field. The cells were cultured in a CO 2 incubator, which was placed in the irradiation room for biological study of the heavy ions in the Heavy Ion Medical Accelerator in Chiba (HIMAC), at the National Institute of Radiological Sciences (NIRS). We measured the absorbed dose, which was determined by a thermoluminescence dosimeter (TLD) and a Si-semiconductor detector, to be 1.4 mGy per day when operating the HIMAC machine. The total population doubling number (tPDN) of the exposed cells reduced to 77–94% of the non-exposed control cells. Furthermore, the shortening of the speed of the telomere length in the exposed cells was much higher than that in the non-exposed cells at the 26th passage, during which period the exposed cells were accumulated at 0.22 Gy. These findings show that the acceleration of senescence in normal human cells occurs by the chronically low-dose irradiation in a heavy-ion radiation field.

Ukrain ®, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation

Purpose : Ukrain ®, an alkaloid thiophosphoric acid derivative of Chelidonium majus L., has demonstrated a promising impact on chemotherapy in a variety of malignancies. The effects of the drug on cell survival, alteration of the cell cycle and induction of apoptosis were examined without and in combination with ionizing radiation (IR). The TP53 status of the cell lines used was also investigated. Materials and methods : Exponentially growing human tumour cell lines MDA-MB-231 (breast), PA-TU-8902 (pancreas), CCL-221 (colorectal), U-138MG (glioblastoma), and human skin and lung fibroblastic cells, HSF1, HSF2 and CCD32-LU were studied by colony assay, flow cytometry (cell-cycle, annexin-V staining for apoptosis) and Western blotting. Ukrain was used in concentrations from 0.1 to 50 μ g ml -1 for 1, 3 and 24 h and radiation as single doses of 1-10Gy. Combined drug-radiation exposure employed 1 μ g ml -1 Ukrain for 24h plus 2-8 Gy. Results : Ukrain cytotoxicity was time- and dose-dependent. The combination of Ukrain plus IR gave enhanced toxicity in CCL-221 and U-138MG cells, but not in MDA-MB-231 and PA-TU-8902 cells. Most strikingly, a radioprotective effect was found in normal human skin and lung fibroblasts. Flow-cytometry analyses supported the differential and cell line-specific cytotoxicity of Ukrain. CCL-221 and U-138MG cells accumulated in G2 after 24-h Ukrain treatment, whereas no alterations were detected in the other tumour cells and normal fibroblasts tested. Western blotting of TP53 demonstrated non-functional overexpression in all tumour cell lines without a ffecting p21. HSF1 presented wild-type TP53 and a p21 response after IR. Flowcytometric analyses of annexin-V staining showed no induction of apoptosis after Ukrain treatment in comparison with untreated controls. Conclusions : Differential effects of Ukrain in modulating radiation toxicity of human cancer cell lines and its protective effect in normal human fibroblasts suggest that this alkaloid may have potential properties for clinical radiochemotherapy.