UV induced bystander signaling leading to apoptosis

Cancer Letters 223 (2005) 275–284 www.elsevier.com/locate/canlet

UV induced bystander signaling leading to apoptosis Gautam Banerjeea,*, Nishma Guptaa, Arun Kapoorb, Govindarajan Ramana,1 a

Cell and Molecular Biology, Environmental Safety Laboratory, Hindustan Lever Research Centre, Mumbai, India b Thapar Institute of Engineering and Technology, Patiala, India Received 12 June 2004; accepted 13 September 2004

Abstract Human keratinocytes (HaCaT) were exposed to UV (ACB) (UVA-350–400 mJ/cm2 and UVB-30 mJ/cm2) which induces apoptosis as evidenced by MTT assay, DNA laddering, Bax and Fas up-regulation. UV induced apoptotic conditioned media (6 h or earlier) did not cause apoptosis in unexposed cells. However, treatment with conditioned medium collected post UV exposure (1 h) induced Bax in unexposed cells as observed by RT-PCR. The induction of cell death was initiated by conditioned medium collected 12 h after UV exposure and the extent of death was increased progressively when conditioned medium collected 24 or 72 h post UV exposure was used. Medium collected 24 h after UV exposure also increased mitochondrial membrane permeability as determined by rhodamine uptake. Conditioned medium induced apoptosis did not involve reactive oxygen species (ROS) unlike UV induced apoptosis indicating that the apoptosis pathway could be different. Interestingly, at high dilution apototic conditioned medium did not induce apoptosis but actually protected cells from UV insult. The role of nerve growth factor (NGF) in UV induced bystander effects are also discussed. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; UV; Keratinocytes; Secreted factors; Protection; Nerve growth factor (NGF); Bystander effect

1. Introduction Cell death or ‘Apoptosis’ is an essential physiological process that occurs in metazoans for their normal growth and development, regulation of cell turnover and as a defense strategy against invading pathogens. Once apoptosis is initiated, it leads to various biochemical and morphological changes, which eventually lead to irreversible degradation of * Corresponding author. Tel.: C91 22 2827 6370; fax: C91 22 2836 3680. E-mail address: [email protected] (G. Banerjee). 1 Present address: Johnson and Johnson PTe Ltd, Singapore.

genomic DNA and fragmentation of dying cell into apoptotic bodies [1,2]. These are recognized and removed by phagocytes, thereby avoiding inappropriate inflammatory responses characteristic of a necrotic response [3]. UV radiation in particular UVB (290–320 nm) from sunlight is one of the most important external stimulus that affects skin by inducing immunosuppression, cancer, premature skin aging, inflammation and cell death [4]. Apoptosis in skin occurs as a result of UV exposure, manifested in form of sunburn cells [5] or during terminal differentiation of keratinocytes in the epidermis [6]. It has been shown that activation of caspase-3 induces apoptosis in UV

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.09.035

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exposed HaCaT cells [7]. Induction of caspases 8, 9 and 3, followed by induction of Fas receptor on membrane by UV exposure is also known [8]. When an area of skin is exposed to UV, apoptosis can be induced in the region that suffers the greatest exposure. Cells surrounding this area would also be damaged partially. We hypothesised that the cells undergoing apoptosis would secrete factors that would induce apoptosis in these partially damaged/ undamaged cells and these soluble factors diffuse further away from the site of maximal injury and would ‘teach’ unexposed cells to overcome the effects of UV exposure. These types of cellular communication is possible through either gap junction (cell to cell) or through irradiated cell conditioned media (ICMM). These types of bystander effects are subjects of intense investigation in radiation biology. These include consideration of radiation induced bystander effects during cell cycle, clastogenic factors, genomic instability and transgenerational effects [9–11]. The proposed factor(s) must have the capacity to stimulate cytokines signaling and/ or free radical generation including reactive oxygen species (ROS) and nitric oxide [12,13]. The aim of the present study was to investigate the role of UV induced bystander signaling effects generated during UV induced apoptosis and their ability in the induction of apoptosis in unexposed cells.

2.1. Cell culturing HaCaT and A431 were maintained in complete DMEM in presence of 10% FCS at 37 8C and 5% CO2. They were subcultured in every 3–4 days using Trypsin-EDTA (0.5 mM EDTA in 0.05% Trypsin). 2.2. UV irradiation For UV irradiation, cells were grown in six well tissue culture plates. For UV exposure the medium was removed from the plate, the cells were washed with PBS and a drop of PBS was added to the plate to keep cells hydrated during the irradiation. Cells were exposed to UV (ACB) using a Wotan lamp. The control cells were treated similarly by the same protocol except radiation. After irradiation, cells were reincubated in DMEM medium overnight. For apoptosis experiments, the dose was (UVA350–400 mJ/cm2 and UVB was 30 mJ/cm2 monitored) using an UV meter (Solar Light Inc, USA). This dose was determined after monitoring a wide range of UV exposure (UVB: 5–90 mJ/cm2). The effect of UVA was also investigated and upto 10 J/cm2 no cell death was seen. Hence all the effects described were due to UVB only and the effects of UVA may be disregarded, as during the exposure the intensity of UVA was 350–400 mJ/cm2. 2.3. Collection of conditioned medium

2. Materials and methods Materials—Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, antibiotics, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TRI reagent were from Sigma, USA. Fetal calf serum (FCS) was from Gibco. All molecular biology reagents were from MBI Fermentas, USA. The PCR primers were from Microsynth, Switzerland. pGem-T easy vector was from Promega, USA. All other materials were of analytical grade. Tissue culture plates were purchased from Nunc. A431 cells were obtained from National Center for Cell Science (NCCS),Pune, India. HaCaT cells was a gift from Dr N.Fusenig, Germany. NGF and anti NGF was a gift from Dr Uma Santhanam, Unilever, UK.

Conditioned medium from irradiated/ non irradiated cells was collected from plates at indicated time points and was centrifuged at 10,000g for 10 min to remove cell debris. The supernatant was collected and used for all assays as described. 2.4. Induction of apoptosis in HaCaT Cell lines using conditioned medium HaCaT cells were exposed to UVACB lamp at a dose of (UVB) 30 mJ/cm2 as described. Fresh medium was added to cells post exposure. The conditioned media were taken after different time intervals for further analysis, i.e. 1, 3, 6, 24 and 72 h post exposure. Conditioned medium from irradiated cells was added to unirradiated cells and incubated

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overnight. Conditioned medium from unirradiated cells was added to freshly plated unirradiated cells and was used as control. Unexposed HaCaT cells were grown on either 96 well plate for MTT assay or six well plates for DNA laddering, RT-PCR assay. For all assays (DNA laddering, NGF related protection/death), cells were incubated with conditioned media from irradiated cells (obtained 16–18 h post radiation) unless otherwise stated. For control experiments, cells were plated and grown simultaneously as those used for radiation. Conditioned media was taken from unirradiated cells at similar time points that of irradiated cells. 2.5. DNA fragmentation DNA was isolated from cells exposed to UV (ACB) and control (unexposed) cells as well as conditioned medium treated unexposed cells by the phenol chloroform extraction method. DNA isolated was incubated with RNAse to remove contaminated RNA. 1.0 mg of each set of DNA was analyzed on a 2% agarose gel and was stained with ethydium bromide. DNA pattern was visualized under UV light. 2.6. MTT assay Extent of cellular death in response to UV (ACB) and conditioned medium collected at various time points post UV exposure was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HaCaT cells were inoculated in 96 well Microtitre plates at the concentration of 1! 105 cells/well. After overnight culture, cells were exposed to single dose of UV (ACB) (UVB30 mJ/cm2). For conditioned medium, 100 ml of medium (from both radiated as well as unirradiated cells) was added to unirradiated cells and incubated overnight. After overnight culture, 10 ml of MTT (5 mg/ml) was added to each well. After 4 h of incubation at 37 8C, the plate was centrifuged at 1000 rpm for 5 0 . The supernatant was removed and precipitate was dissolved in 100 ml of acidic isopropanol. The O.D of the resulting solution was measured spectrophotometrically at 540 nm.

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2.7. cDNA preparation and PCR RNA was isolated from unirradiated cells and UV radiated cells as well as cells treated with conditioned media collected at various time points post UV radiation using TRI reagent as per instruction manual. cDNA was prepared using the reverse transcriptase kit as per instruction manual (MBI Fermentas). Presence of Bax and Fas was carried out by PCR using sequence specific primers. b-actin was used as internal control to ensure that equal amounts of RNA was used. PCR was conducted in 1!PCR buffer with 2.5 mM MgCl2 supplemented with 0.2 mM dNTP’s, 20 pmol sense and antisense primers for Fas, Bax, b-actin, 1 ml cDNA, 1 unit of Taq DNA polymerase in a 20 ml reaction mixture. PCR profile consisted of 35 cycles of 94 8C for 60 s, 57 8C for 90 s, 72 8C for 2 min and final extension at 72 8C for 10 min. PCR products were analyzed on a 2% agarose gel and stained with ethidium bromide. The sequences of the primers are given below Bax Forward 5 0 CAGCTCTGAGCAGATC-3 0 Reverse 5 0 GCCCATCTTCTTCTTC-3 0 Fas Forward 5 0 CCTCCTACCTCTGGTTCTTACGTC-3 0 Reverse 5 0 GCCATGTCCTTCATCACACAATC-3 0 b-actin Forward 5 0 AGCGGGAAATCGTGCGTG-3 0 Reverse 5 0 CAGGGTACATGGTGGTGCC-3 0 2.8. Protection assayHaCaT cells were plated in 96 well plates and incubated overnight. Adherent cells were incubated with 1:100 dilution of conditioned medium (obtained 16 h post UV exposure) overnight. The following day the medium was removed, washed with PBS. The cells were exposed to UV (ACB) (30 mJ/cm2) and incubated overnight. MTT assay was done as described earlier.

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2.9. Nerve growth factor induced protection and killing HaCaT cells were plated in 96 wells plates and incubated overnight as described before. These adherent cells were incubated with various concentrations of nerve growth factor (NGF) from 10 mg/ml to 100 ng/ml for overnight. After overnight incubation, MTT assay was done to monitor NGF induced cell death or survival. In a separate sets of experiments the adherent cells were incubated with 10 mg/ml of NGF as a function of time. After the stipulated time of incubation, MTT assay was done as before. In a separate experiment HaCaT cells were incubated with 16 h post UV exposure conditioned medium in presence and absence of anti NGF (5 mg/ml). After overnight incubation, MTT assay was done as described before.

DNA was isolated and analysed on a 2% agarose gel. Distinct DNA laddering was observed in response to the UV doses. The increase in the intensity of the low molecular weight bands in the ladder was proportional to the increase in the UV dose. In control HaCaT cells, an intact high molecular weight band was observed. The effect of apoptotic conditioned medium on DNA of unirradiated HaCaT cells was analysed. The result is shown in Fig. 2. DNA isolated from unexposed cells treated with 12 and 24 h conditioned media post UV exposure showed DNA damage to a significant extent while the conditioned medium from unirradiated cells withdrawn at similar time points had no effect.

3. Results 3.1. Induction of apoptosis by secreted factors The MTT assay was done to identify the cell survival in unexposed cells after exposure to apoptotic-conditioned medium. Fig. 1 shows that apoptotic death was initiated by conditioned media harvested post 12 h of UV exposure. Early conditioned medium (less than 6 h) did not cause any apoptotic cell death. Post 12 h conditioned medium induced apoptotic death of unirradiated HaCaT and the survival of cells decreased with conditioned media collected later than 12 h. Conditioned media collected from cells after 24 h incubation caused 50–60% death while that with 72 h medium caused only 70% death (30% survival). It is possible that 72 h post exposure medium caused more death due to depletion of nutrients in the medium. Removal of media 3 h after exposure and addition of fresh media did not have any effect on the percentage of dead cells. 3.2. DNA laddering in response to secreted factors Apoptosis was induced in HaCaT keratinocytes using single UV dose (UVB-30 mJ/cm2). Cells were harvested 24 h after UV exposure and subsequently

Fig. 1. (a) Effect of conditioned medium from UV exposed HaCaT cells (obtained 0–72 h post exposure) on normal HaCaT cells. Results are expressed as meanGSD of three different experiments. Unexposed HaCaT cells were incubated in post UV exposure spent medium for 16 h. Survival was determined by MTT assay. The survival index was compared to untreated control cells (100%) (cells treated with conditioned medium from unexposed cells). (b) A representative experiment as described in (a). The results are shown in terms of change of optical density (O.D) using apoptotic conditioned medium compared to normal conditioned medium.

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Fig. 2. Effect of UV and post UV exposed conditioned medium on unexposed HaCaT cells. The figure shows effect on integrity of chromosomal DNA. Lane-1: 1 kb DNA ladder, lane-2: unexposed cells, Lanes 3–5: UV exposed cells for 30, 60, 90 mJ/cm2, respectively. Lane-6: unexposed cells. Lane-7: 12 h conditioned medium from unexposed cells Lane-8: 12 h conditioned medium from UV exposed cells (30 mJ/cm2). Lane-9: 24 h conditioned medium from unexposed cells. Lane-10: 24 h conditioned medium from UV exposed cells (30 mJ/cm2).

3.3. Presence of apoptotic markers in UV exposed cells 3.3.1. Presence of Bax and Fas The expression of certain apoptotic markers was analysed in UV radiated HaCaT cells. Regulation of transcript of Bax gene (600 bp band) was seen in UV radiated HaCaT cells. No band corresponding to mRNA of Bax gene was observed in unirradiated HaCaT cells. The effect of apoptosis induced conditioned medium on Bax gene was also investigated. It was found that Bax was induced in presence of post UV exposed conditioned medium (collected from cells post 12 and 24 h radiation) while the presence of Bax was not detected in unirradiated cells. This gene was detected at as early as with 1 h post radiated condition medium while the intensity increases with conditioned media collected at later time points. The result is shown in Fig. 3. The intensity of the bands was similar at 12 and 24 h post exposure. We have also investigated the effect of conditioned medium from HaCaT cells on another keratinocyte cell line, A431. We could not detect the presence of Bax in unirradiated cells but Bax mRNA was transcribed in cells exposed to post UV conditioned medium (data not shown). Fas gene was shown to be expressed constitutively in unexposed HaCaT cells and was up-regulated upon exposure. A semi-quantitative PCR was performed to check for the expression level of Fas. At 1:200

dilution there was no band corresponding to Fas in control cells whereas Fas expression was still present in the UV exposed cells (data not shown). Thus distinct upregulation of Fas gene was observed in UV induced apoptotic cells. The presence of Fas was also investigated using the same conditioned medium as

Fig. 3. Effect of conditioned medium on Bax expression in unexposed cells. (a) Bax. (b) b -actin, Lane-1: unexposed cells, Lane-2: UV exposed cells (30 mJ/cm2), Lane-3: unexposed cells. Lane-4: 1 h conditioned medium from exposed cells, Lane-5: 12 h conditioned medium from exposed cells, Lane-6: 24 h conditioned medium from exposed cells.

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death prevailed. The result is shown on Table 2a. NGF did not cause cell death at the range of 100 ng/ ml to 1.0 mg/ml. Instead, cells proliferated more than control. Effect of NGF was also investigated as a function of time. NGF, at a concentration of 10 mg/ml, induced cell death at 24 h of incubation while below 12 h there were no cell death. The results are shown on Table 2b. In presence of anti-NGF, conditioned medium induced apoptotic death was not seen while the conditioned medium alone (in absence of anti NGF) could induce apoptotic death. The result is shown on Table 2c.

4. Discussion

Fig. 4. Effect of conditioned medium on expression of Fas in unexposed cells. (a) Fas (b) b-actin, Lane-1; Unexposed cells. Lane-2: 1 h conditioned medium from exposed cells, Lane-3: 12 h conditioned medium from exposed cells, Lane-4: 24 h conditioned medium from exposed cells.

described for Bax analysis and it was observed that expression of Fas was upregulated in conditioned medium treated cells while some basal level expression was detected in normal unexposed cells. The result is shown in Fig. 4. The intensity at 24 h post exposure was more compared to 1 or 12 h medium. 3.3.2. Protection by conditioned medium at high dilutionThe results in Table 1 show that apoptotic death was induced when cells were exposed to UV. The unirradiated cells are unaffected when incubated with diluted conditioned media (1:100) in absence of UV exposure. UV exposure to cells previously incubated with diluted conditioned media inhibits apoptosis induction. The inhibition of apoptosis by conditioned media was significantly higher compared to untreated radiated cells. 3.3.3. Effect of NGF and anti NGF NGF at low concentration (100 ng/ml) induced cell survival while at higher concentrations (5 mg/ml) cell

Cells possess complex systems including receptors, kinases, GTP binding proteins and several other small adapter molecules that enable them to establish an efficient cell–cell signaling mechanism [14]. Molecules secreted by a cell can be carried far away to act on distant target or can act locally by affecting cells in close environment. These bystander effects are mainly consequences of various autocrine/paracrine signaling pathway [15]. Although gap junction intercellular communication (GJIC) is an important pathway for the production of bystander responses, it is not always required. It has been found that some signaling factors including ROS and TGF can be produced from irradiated cells and play important roles in medium mediated bystander effects [16,17]. Further, the involvement of iNOS and cNOS has also been recently identified [13,18]. The apoptotic process plays a critical role in the implementation of physiological and pathological cell death. It is controlled by a conserved genetic program that is activated in dying cells. This machinery includes caspases, Apaf-1 like adapter molecules and Bcl-2 family members. The pro and anti apoptotic members of Bcl-2 family governs the fate of the cells. This death induction is initiated by translocation of phosphatidylserine (PS) molecules followed by DNA damage and induction of various pro-apoptotic members of BCl-2 family [19]. We have shown that apoptosis is caused by UVACB at a dose of as low as 20 mJ of UVB. At higher doses the extent of cell death increased as indicated by MTT assay. Beyond 60 mJ, necrosis predominates over apoptosis (data not shown).

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Table 1 Inhibition of UV induced apoptosis by diluted spent media (16 h post UV exposure) treated unexposed HaCaT cells Unexposed cells

Exposed cells

Normal medium O.D Survival (%)

Spent medium (diluted 1:100)

a,c

Normal medium

0.558G0.0032 107.7G5.7

0.518G0.010 100G1.9

Spent medium (diluted 1:100)

a,b

0.481G0.011b,c 92.8G2.2

0.425G0.011 82.0G2.6

HaCaT cells were plated in 96 well plate and incubated overnight. The adherent cells were incubated with 1:100 dilution of conditioned medium (obtained 16 h post UV exposure K30 mJ/cm2) overnight. The following day these cells were exposed to UV (ACB) for 30 mJ/cm2 and incubated overnight. MTT assay was done as described earlier. The values indicate the optical densities (O.D) of the MTT assay. a r!0.0001 (Comparison of column 1 and 3). b r! 0.001 (Comparison of column 3 and 4). c r!0.01 (Comparison of column 1 and 4). The result is expressed as mean G SE of five different sets of experiments (nZ16–32 in each set).

The extent of DNA laddering was also increased at higher UV dose as an indicative of DNA damage. The role of Bax, a pro-apoptotic member of BCl-2 family was investigated in apoptotic death, induced by UVB as well as UV radiated conditioned media. Bax was induced at mRNA level by UV at apoptotic dose but it was not detected in unexposed cells. It is known that Bax up-regulation caused apoptotic death in fibroblasts [20], keratinocytes [21] by altering the binding of anti-apoptotic members of Bcl-2 family. Our results showed that the conditioned medium from irradiated cells (12 h incubation post exposure) caused apoptotic death of HaCaT while early conditioned medium (6 h or less) did not induce any apoptotic death. It is possible that the concentrations of death initiators are low at early time points and increase at 6 h and beyond. It is also observed that post 24 h conditioned medium causes more apoptotic death than post 12 h conditioned medium and maximum apoptotic death is caused by post 72 h conditioned medium. DNA laddering was also clearly observed in cells incubated in 12 and 24 h conditioned medium. Although induced expression of proapoptotic Bax gene was seen in cells incubated in 1 h conditioned medium, indicating the potential of 1 h

conditioned medium in elevated expression of pro apoptotic gene. It is possible that at 1 h sufficient amount of death initiator(s) may not be formed or level of expression may not be sufficient to cause apoptosis in normal cells using conditioned media. Hence the death machinery initiated by post 12 h conditioned medium where the concentrations of death initiators or pro-apoptotic members are sufficiently high to induce apoptosis. Expression of Fas was also observed in unexposed cells, showing its expression at basal level while post 12 and 24 h conditioned medium induced the up regulation of Fas in unirradiated cells. Our results suggest that 12 and 24 h conditioned medium can bring about upregulation of Fas and induction of Bax (as is done by UV exposure) and also the death of normal unexposed cells which may be due to some secreted molecules in the conditioned medium. In a recent study it is reported that medium from keratinocytes exposed to gamma radiation can also induce apoptosis in unirradiated cells [22]. It is mentioned that conditioned media from gamma irradiated cells induced CaC2 mobilisation as well as release of reactive oxygen species (ROS) although we could not detect the presence of ROS using death

Table 2a Effect of NGF on apoptosis and proliferation Control O.D Survival (%)

0.47G0.08 100G8.2

NGF (mg/ml) 10.0

5.0

1.0

0.5

0.1

0.39G0.04a 82.9G5.2

0.44G0.08 93.6G8.2

0.53aG0.04 112.7G3.8

0.53G0.07 112.7G6.6

0.53G0.07 112.7G5.8

HaCaT cells were plated in 96 well plate and incubated overnight. The adherent cells were incubated with various concentrations of nerve growth factor (NGF). After overnight incubation, MTT assay was done to monitor NGF induced cell death or survival. The result is expressed as mean G SE of two different sets of experiments (nZ32 in each set). ar!0.001 compared to control.

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Table 2b Effect of incubation time of NGF on apoptosis and proliferation 1h

12 h

Control

NGF a

0.701G0.066

24 h

Control a

0.768G0.08

NGF b

0.914G0.04

Control b

0.892G0.036

NGF c

0.959G0.07

0.81G0.073c

HaCaT cells were plated in 96 well plate and incubated overnight. The adherent cells were incubated with various concentrations of nerve growth factor (NGF). After overnight incubation, MTT assay was done to monitor NGF induced cell death or survival. The result is expressed as a representative set of two different experiments. ar!0.1 compared to control; bnot significant; cr!0.001 compared to control.

inducing conditioned media. It is not known whether during apoptosis induced by either UV or gamma rays, the molecules secreted are same or different. The reason of conferring protection or resistance to UV is also not clear. From our results we hypothesize that the conditioned media contains some soluble factors which are operating through binding of various receptors (responsible for both survival and death) present on the cell surface. At higher dilution (low concentration), the molecule binds to high affinity receptor involved in survival. At lower dilution (high concentration), once the high affinity receptor is blocked, the same molecule binds to low affinity receptor responsible for inducing the death machinery. It is known that [23] keratinocytes secretes growth factors (Nerve growth factor or NGF) which possess two specific receptors (p75) and (trkA) with different affinities on the cell surface. The high affinity receptor (trkA) helps in proliferating cells while blocking of this receptor induces apoptosis [24,25]. This high affinity receptor protect cells from UV-B induced apoptosis by maintaining a constant level of Bcl-2 [23]. From our data it can be concluded that, exposure of HaCaT cells to early apoptotic conditioned medium induces proliferation whereas apoptosis was induced with medium at later time points. It is possible that

the concentration of NGF (and other growth factors) was very low at early time points hence only high affinity receptors were activated. As a result cells were proliferating more than control. At later time points, when the concentration was high, low affinity receptors were also involved and apoptosis prevailed. These pathways are mutually exclusive and may depend upon the concentration of the mediators. We have seen that amount of NGF was more in apoptotic conditioned medium than normal conditioned medium taken at same time points after plating (data not shown). The anti NGF antibody removed the NGF hence cell survived in presence of apoptotic conditioned medium. The exact mechanism is not known and is being investigated. In a recent report, it has been shown that NGF regulated apoptosis through NADE (p75 NTRassociated cell death executor by interacting with the cell death domain of p75 NTR (p75 neutrophin receptor) [26]. In a recent report, involvement of TNFRp55 has also been implicated [27]. In the case of conditioned media (UV radiated) treated cells, killing of unirradiated cells has been related to the secretion of a protein like signal able to initiate apoptosis. IL-8, TNF-a are some of the probable candidates involved in radiation induced by stander effect but not been confirmed previously [16].

Table 2c Effect of NGF and anti NGF on apoptosis

O.D Survival (%)

Control

NGF (10 mg/ml)

NGF (100 ng/ml)

Conditioned media (undiluted)

Conditioned media Canti NGF

0.251G0.006 100G2.3

0.219G0.007a 87.6G3.1

0.268G0.003b 106.7G1.1

0.217G0.007a 86.4G3.2

0.30G0.004a 120G1.3

HaCaT cells were plated in 96 well plate and incubated overnight. The next day adherent cells were incubated with nerve growth factor (NGF) at 10 mg/ ml and 100 ng/ml. After overnight incubation, MTT assay was done to monitor NGF induced cell death or survival. In a separate experiment HaCaT were incubated with 16 h post UV exposure conditioned medium in presence and absence of anti NGF (5 mg/ml). After overnight incubation, MTT assay was done as described before. The values indicate the optical densities (O.D) of the MTT assay. The result is expressed as meanGSE of two different sets of experiments (nZ32 in each set). The values for anti NGF on unexposed cells was 0.24G0.008. a r!0.001 compared to control; br!0.01 compared to control.

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The possibility of bystander signals induced cell death as well as oncogenic transformation has been proposed using various techniques [28–30]. It has been shown that bystander component of radiation response can increase with dose and can be observed at high doses as well as low doses which is correlated with our findings. UV radiation is known to induce apoptosis as well as carcinogenesis through DNA mutation. If non apoptotic or resistant cells clonaly expand to carcinogenesis which does not result directly from DNA mutation, then this carcinogenic process may not be related to radiation dose. Hence a ‘bifurcation’ model can be proposed that could accommodate both beneficial and harmful effects of radiation through signaling molecules. We have seen by RAPD, three unique bands in cells treated with undiluted conditioned medium and we have cloned these DNA. One of these genes has been demonstrated to be up-regulated during UV induced apoptosis on HaCaT but it has no sequence similarity with any known protein. It is possible those genes regulated (up/ down) by conditioned medium will be similar to those regulated during apoptosis/ survival as the genes we are investigating are originated from target cells [31]. Hence, at this point it is difficult to predict the regulation of entire gene profile of the target cells unless the identification and cloning of those genes followed by expression studies are complete. We are in the process of cloning and characterising the genes regulated in the target cells.

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