In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice.

UV irradiation has been shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in cell culture; however, only limited studies have been described in vivo. UV light has been categorized as UV-A (400 to 315 nm), -B (315 to 280 nm), or -C (less than 280 nm); the longer wavelengths are less harmful but more penetrative. Highly penetrative UV-A radiation constitutes the vast majority of UV sunlight reaching the earth's surface but is normally harmless. UV-B irradiation is more harmful but less prevalent than UV-A. In this report, the HIV-1 LTR-luciferase gene in the skin of transgenic mice was markedly activated when exposed to UV-B irradiation. The LTR in the skin of transgenic mice pretreated topically with a photosensitizing agent (psoralen) was also activated to similar levels when exposed to UV-A light. A 2-h exposure to sunlight activated the LTR in skin treated with psoralen, whereas the LTR in skin not treated with psoralen was activated after 7 h of sunlight exposure. The HIV-1 LTR-beta-galactosidase reporter gene was preferentially activated by UV-B irradiation in a small population of epidermal cells. The transgenic mouse models carrying HIV-1 LTR-luciferase and LTR-beta-galactosidase reporter genes have been used to demonstrate the in vivo UV-induced activation of the LTR and might be used to evaluate other environmental factors or pharmacologic substances that might potentially activate the HIV-1 LTR in vivo.     

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures.

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.     

Analysis of substances issuing from Zajdela ascitic hepatoma cells following exposure to UV radiation of different wavelength. II. Proton release]

The release of protein from the Zaidela ascitic hepatoma cells following irradiation with physiological doses of short-wave (254 nm) and long-wave (300--380 nm) UV light (far and near UV radiation) has been investigated. The amount of protein increases with dose making, upon the maximal radiation damage, 180 and 2 per cent of the protein against, resp., the protein amount releasing from non-treated cells and the total protein of the intact cell. The far UV light is by one order more efficient than the near UV light. Irradiation of cells with the former and the latter results in the release of high and low molecular proteins, resp. The near UV irradiation brings about heavier releasing of proteins than does the far UV light.     

Ultraviolet Rays Promote Development of Photosystem II Photochemical Activity and Accumulation of Phenolic Compounds in the Tea Callus Culture (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Effect of UV-B rays (280–320 nm) on photosynthetic electron transport and production of phenolic compounds in tea (Camellia sinensis L.) callus culture grown in white light was investigated. When white light was supplemented with UV radiation, the culture growth was retarded and morphological characteristics were modified. These conditions promoted the formation of chlorophyll-bearing cells and altered the ability of cultured cells to accumulate phenolic compounds, including flavans specific to Camellia sinensis. By the end of the culturing cycle (on the 45th day), the total content of phenolic compounds in the culture grown under supplementary UV irradiation was almost 1.5 times higher than in the control culture. The UV rays greatly stimulated photosystem II (PSII) activity in phototrophic cells of the callus culture, which was indicated by a large increase in the ratio of variable chlorophyll fluorescence to maximal fluorescence. This ratio was as low as 0.19 in cells cultured in white light and increased to 0.53 in the cell culture grown under white and UV light. The kinetics of dark relaxation of chlorophyll variable fluorescence, related to reoxidation of PSII primary acceptor, contained either two or three components, depending on the absence or presence of UV radiation, respectively. An artificial electron acceptor of PSI, methyl viologen modified the kinetics of dark decay of chlorophyll variable fluorescence in a characteristic manner, implying that photosynthetic electron transport was mediated by PSI and PSII in both treatments (culturing in white light with and without UV-B). It is concluded that stimulatory effect of UV rays on the parameters examined in phototrophic regions of Camellia tissue culture is determined by photoexcitation of a regulatory pigment that absorbs quanta in blue and long-wave UV spectral regions.     

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5-7 DPC per 10(10) dalton were induced in these cells by either 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light (PRL) or 10 kJ/m2 of sunlamp UV greater than 295 nm. The irradiated cells were then incubated for 0-24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV greater than 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV greater than 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.     

Analysis of substances issuing from Zajdela ascitic hepatoma cells after exposure to UV radiation of different wave-lengths. I. Relationship to dose]

The release of substances from the Zaidela ascitic hepatoma cells after irradiation with physiological doses of short-wave (254 nm) and long-wave (300-380 nm) UV light (far and near UV light) has been studied spectrophotometrically. Within the range of 200-520 nm, the absorption spectra of releasing substances show maxima at 215 and 260 nm and are identical to spectra of non-irradiated cells. The amount of substances increases with dose making up, at the maximal alteration, 180-220%, of the amount releasing from non-irradiated cells. Irradiation with far UV light exceeds by one order that with near UV light. The effect of minimum doses is opposite to the action of high doses: the release of substances from irradiated cells is much less.     

Perception of solar UV radiation by plants: photoreceptors and mechanisms

About 95% of the ultraviolet (UV) photons reaching the Earth’s surface are UV-A (315–400 nm) photons. Plant responses to UV-A radiation have been less frequently studied than those to UV-B (280–315 nm) radiation. Most previous studies on UV-A radiation have used an unrealistic balance between UV-A, UV-B, and photosynthetically active radiation (PAR). Consequently, results from these studies are difficult to interpret from an ecological perspective, leaving an important gap in our understanding of the perception of solar UV radiation by plants. Previously, it was assumed UV-A/blue photoreceptors, cryptochromes and phototropins mediated photomorphogenic responses to UV-A radiation and “UV-B photoreceptor” UV RESISTANCE LOCUS 8 (UVR8) to UV-B radiation. However, our understanding of how UV-A radiation is perceived by plants has recently improved. Experiments using a realistic balance between UV-B, UV-A, and PAR have demonstrated that UVR8 can play a major role in the perception of both UV-B and short-wavelength UV-A (UV-A_sw, 315 to ∼350 nm) radiation. These experiments also showed that UVR8 and cryptochromes jointly regulate gene expression through interactions that alter the relative sensitivity to UV-B, UV-A, and blue wavelengths. Negative feedback loops on the action of these photoreceptors can arise from gene expression, signaling crosstalk, and absorption of UV photons by phenolic metabolites. These interactions explain why exposure to blue light modulates photomorphogenic responses to UV-B and UV-A_sw radiation. Future studies will need to distinguish between short and long wavelengths of UV-A radiation and to consider UVR8’s role as a UV-B/UV-A_sw photoreceptor in sunlight.

In sunlight, UVR8 mediates the perception of both UV-B and short-wavelength UV-A radiation with its sensitivity moderated by blue light perceived through cryptochromes.     

UV‐A/BLUE LIGHT–INDUCED REACTIVATION OF SPORE GERMINATION IN UV‐B IRRADIATED ULVA PERTUSA (CHLOROPHYTA)1

Recent reduction in the ozone shield due to manufactured chlorofluorocarbons raised considerable interest in the ecological and physiological consequences of UV‐B radiation (λ=280–315 nm) in macroalgae. However, early life stages of macroalgae have received little attention in regard to their UV‐B sensitivity and UV‐B defensive mechanisms. Germination of UV‐B irradiated spores of the intertidal green alga Ulva pertusa Kjellman was significantly lower than in unexposed controls, and the degree of reduction correlated with the UV doses. After exposure to moderate levels of UV‐B irradiation, subsequent exposure to visible light caused differential germination in an irradiance‐ and wavelength‐dependent manner. Significantly higher germination was found at higher photon irradiances and in blue light compared with white and red light. The action spectrum for photoreactivation of germination in UV‐B irradiated U. pertusa spores shows a major peak at 435 nm with a smaller but significant peak at 385 nm. When exposed to December sunlight, the germination percentage of U. pertusa spores exposed to 1 h of solar radiation reached 100% regardless of the irradiation treatment conditions. After a 2‐h exposure to sunlight, however, there was complete inhibition of germination in PAR+UV‐A+UV‐B in contrast to 100% germination in PAR or PAR+UV‐A. In addition to mat‐forming characteristics that would act as a selective UV‐B filter for settled spores under the parental canopy, light‐driven repair of germination after UV‐B exposure could explain successful continuation of U. pertusa spore germination in intertidal settings possibly affected by intense solar UV‐B radiation.     

Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2). Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.     

Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.     

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.     

Growth inhibition of mammalian cells by synthetic and natural photosensitising agents

Amammalian cell line, J774, was susceptible to both synthetic and natural photosensitising agents after irradiation with long-wave ultraviolet light. Both UV-A light and psoralen did not affect cell growth individually; a reduction invisual confluency was achieved only when psoralen and UV-A light were used in combination. The maximum visual confluency decreased by 55% when 50 ppm psoralen was added to a growing culture and irradiated with UV light for 3 min. Decreasing the UV-A exposure times from 3min to 3 s did not greatly affect the maximum total visual confluence reached using different synthetic psoralen concentrations, but did affect the rate at which cell death occurred. The 3 min exposure time resulted in a rapid decrease in cell numbers in comparison to 3s exposure time. Synthetic psoralen was found to have an increasing photosensitising activity with increasing concentration using a logarithmic shift between 0.5 ppm and 50 ppm. A visual confluency of 45 % was achieved using concentrations of 50 ppm psoralen, and 70% visual confluency using 0.5 ppm. Natural mixtures of furanocoumarins containing psoralens, obtained from two separate parsley sources, were found to have greater efficacy at inhibiting the growth cycle of the cells when compared to the synthetic psoralen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxidant stress distressed

Summary

The range of photon energies in solar radiation and the diverse cell and molecular targets in skin allow for participation of oxygen radicals and oxidative stress at several levels in the development of skin cancer: DNA damage and mutation, membrane damage, and intracellular signalling. The intense UVA component of sunlight (315–400 nm) is of particular interest because of deep penetration, generation of oxidative damage and having a mutational spectrum which overlaps that of the more carcinogenic UVB (280–315 nm). Many UV-induced mutagenic and signalling events are now understood at the molecular level, and significant protection from UV carcinogenesis has been obtained with antioxidants in experimental animals. There is little evidence to suggest, however, that similar results have been achieved in humans although the converse effect has been established, of elevated skin cancer risk following simultaneous exposure to sunlight and precursors of the pro-oxidant paraquat. The present difficulty in translating these findings to prevent human skin cancer may arise from deficiencies in the models used and incomplete information about the specific responses of the target cells relevant to solar UV.     

Changes in gene expression by 193- and 248-nm excimer laser radiation in cultured human fibroblasts.

Tissue ablation by ultraviolet excimer lasers results in exposure of viable cells to subablative doses of radiation. To understand the potential biological consequences better, we have studied changes in gene expression in cultured human skin fibroblasts exposed to either 193- or 248-nm laser light. Northern blot analyses revealed that both treatments up-regulate a common set of genes, including interstitial collagenase, tissue inhibitor of metalloprotease, metallothionein, and the proto-oncogene c-fos. Dose-response and kinetic studies of collagenase induction by 193-nm radiation showed a maximal effect with 60 J/m2 and at approximately 24 h. The induction was still persistent 96 h later. In addition to the commonly affected genes, known to be activated also by conventional UV light (254 nm) and tumor-promoting phorbol esters, other genes were found to be selectively induced by the 193-nm radiation. The heat-shock hsp70 mRNA, undetectable in controls and in cultures irradiated at 248 nm, was transiently induced 8 h after exposure to 193-nm radiation. Furthermore, a selective up-regulation of collagen type I expression was observed. The results indicate that the 193- and 248-nm radiations by excimer lasers elicit specific and different cellular responses, in addition to an overlapping pathway of gene activation common also to UV radiation by germicidal lamps. The laser-induced genes could serve as molecular markers in evaluating cell injury in situ.     

A simple method for the comparative evaluation of the rate of reparative synthesis in cultured mammalian cells]

A sensitive method for estimating the rate of repair synthesis is proposed. The method is based on the capacity of long-wave UV light to inactivate cells having minor amounts of bromodeoxyuridine (BUdR) incorporated. This treatment is absolutely nontoxic for cells lacking BUdR. The efficacy of the treatment is achieved by sensitizing cells with a fluorescent dye Hoechst 33258. The proposed method has proved to be effective in studying the repair synthesis in HeLa cells after exposure to short-wave UV light and gamma rays.     

Induction of a Futile Embden-Meyerhof-Parnas Pathway in Deinococcus radiodurans by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO₂. Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.     

Ultraviolet light induces the sustained unscheduled expression of cyclin E in the absence of functional p53

Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G1 phase of the cell cycle and peaks near the G1/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G1/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.     

Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms

We describe a detailed procedure to create photolabile, polyethylene glycol (PEG)-based hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in two-dimensional (2D) or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. In this study, we provide coupling procedures to PEG-bis-amine to form a photodegradable cross-linker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable cross-linker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Degradation is readily achieved with cytocompatible wavelengths of low-intensity flood irradiation (365-420 nm) in minutes or with high-intensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of photodegradable monomers take approximately 2 weeks, but the process can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of sterile solutions for hydrogel fabrication takes hours, whereas the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. Although other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the presence of cells down to the micrometer scale.     

Genome-wide gene expression analysis in cancer cells reveals 3D growth to affect ECM and processes associated with cell adhesion but not DNA repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact.