In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings.

For gene expression analysis, Jurkat cells were treated with HAL-PDT, and total RNA was harvested from triplicate samples of non-treated and treated cells at 1, 2 and 4 h after HAL-PDT. Gene expression levels in HAL-PDT treated cells relative to non-treated cells were determined by oligo microarrays that contained a total of 44,308 oligos targeting human genes. The Venn diagram shows that 146 probes (103 unique Entrez gene IDs) exhibited significant changes (p < 0.05) 1 h after HAL-PDT treatment (Fig. (Fig.2A).2A). The number of significantly altered probes/genes was reduced to 68 probes (53 unique Entrez gene IDs) at 2 h post treatment, with a subsequent large increase at 4 h, with 15,762 probes (10,373 unique Entrez gene IDs). The large number of genes with altered expression at the 4 h time point might reflect a high degree of disturbance in transcriptome within the pro-apoptotic cell. Therefore these genes will be discussed separately as late response genes.

Contrary to early responses, the number of altered genes/probes increased sharply to a total of 15,762 probes (10,373 unique Entrez gene IDs) at 4 h, clearly displaying the massive disturbances in the transcriptome within the pro-apoptotic cells. The proportion of genes with up-regulated expression decreased to 41.1% at 4 h, compared to 82.9% of up-regulated genes at early response. Since the destructive PDT dose likely causes high levels of oxidative stress, we cannot neglect that the high number of altered genes might likely indicate random transcriptome changes, caused by direct oxidation of non-specific mRNAs [63]. RNAs are reported to be more sensitive to photodynamic degradation than DNA [56], especially since most RNAs are cytoplasmic and exposed to higher ROS concentrations. In the case of RNA molecules themselves are photooxidative targets, one could expect that the mRNA transcripts would be damaged randomly and not bind to the specific probes on microarrays. As a result, the expression of genes will be measured as down regulated compare to untreated cells. To explore whether the observed RNA down-regulation at 4 h was direct and non-specific, or targeted, we performed a Gene Ontology and KEGG analysis. We observed that down-regulated genes contributed significantly in GO enriched groups within the total of differentially expressed genes (Table 1, 2). While up-regulated genes scored in other GO groups, a number of enriched extra-nuclear processes, especially mitochondrial, were present when down-regulated genes were scored. However, since HAL, the mitochondrial localizing sensitizer was used in the study [43,64-67], the direct effect on mitochondria was expected. Furthermore, enriched GO processes of up-regulated genes included both nuclear and extra-nuclear processes, such cytoskeleton, junctions and adhesion. Notably, because of the direct PDT effect to cytoskeleton, well-known house keeping proteins such actin or tubulin that is widely used for the quantitation of western data would not be representative controls. Thus, for western blots, the total protein content was used as a measure, by staining the gels after electrophoresis and signals normalized against total protein content. Interestingly, genes encoded proteins in the porphyrin metabolism were stimulated, indicating cellular response to excessive porphyrin production after the transfection with HAL, which is a heme precursor. Among these genes, 7 different UDP glucuronosyltransferase 1 and 2 family genes were induced. These are of major importance in heme degradation [68,69]. The gene ontology analysis suggested that the cell commitment to apoptosis was directly reflected in the transcriptome in a specific manner, and not random changes.

In our experiments, we observed that HAL-PDT resulted in expression changes in the spliceosome complex. A total of 72 probes (44 unique Entrez gene IDs) showed significant changes at 4 h (Table 2), while expression of 39 genes were down-regulated (Fig. (Fig.8).8). None of the splicing factors were significantly altered at early response time points. Alternative splicing is tightly regulated in a tissue-specific and developmental-stage-specific manner [30,71]. Since the microarrays included constitutive and alternative splice variant probes, we examined how many genes were alternatively spliced in response to HAL-PDT (Table 3). A total list of altered splice variants is presented [see Additional file 1]. Interestingly, of 32 genes with altered expression, 24 alternatively spliced variants were down-regulated already after 1 h. Notably, we did not detect changes in the transcriptional level of splice factors at early response times. However, stress-induced signaling mechanisms that regulate pre-mRNA splicing in vivo, by influencing the subcellular distribution of splicing factors, have been described [72]. This type of the regulation will not be detected by monitoring the transcription of splice factors. The increased proportion of down-regulated alternatively spliced variants remained similar (70.6 and 69.2%) at 2 and 4 h, most likely signifying a dysfunction of the splicing machinery following treatment. Furthermore, when the low fold changed variants [1–1.99] were taken into account, the percent of down-regulated splice variants increased to 90, 80 and 65.6, at 1, 2 and 4 h post HAL-PDT, respectively. In contrast, in the groups of the alternative splice variants altered by higher fold change [≥2], the proportion of up- and down-regulated genes were close to equal (Table 3). This indicated nonrandom (caused by the dysfunction of splicing machinery) changes in alternative splicing. Thus, we used GO to uncover whether the genes with alternatively spliced variants could be mapped to particular GO groups/altered processes (see Table 4 for GO biological processes of altered splice variants at 4 h). Interestingly, the GO processes of altered alternative splice variants were relatively similar to the previously scored GO groups, and included different metabolic processes, apoptosis, cell cycle as well as transcription. Interestingly, 5 genes had up-regulated, and 37 had down-regulated alternative splice variants of the genes that had transcription factor activity. Notably, alternative splicing of pre-mRNAs encoding transcription factors is one of the common mechanisms for generating the complexity and diversity of gene regulation [73]. One of the genes was identified as activating transcription factor 3 (ATF3), that had an induced alternative splice variant at all three studies time points, while the constitutive, full-length variant was low expressed and did not change with the treatment. ATF3 is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family of transcription factors [74]. ATF3 plays a role in determining cell fate and has been shown to generate a variety of alternatively spliced isoforms in response to stress [75-78]. It was demonstrated that ATF3 (deltaZip2) isoform, but not full-length ATF3, sensitizes cells to apoptotic cell death, partly by suppressing the expression of the NF-kappaB-dependent anti-apoptotic genes cIAP2 and XIAP. ATF3 (deltaZip2) has further been shown to bind directly to the p65 subunit of NF-kapaB and down-regulate the CBP/p300 recruitment complex [78]. The sequence analysis of the detected alternative splice variant revealed that the alternative splice variant induced by HAL-PDT was the ATF3 isoform (deltaZip2), demonstrating a direct cell fate control through alternative splicing in HAL-PDT response. Thus, the results stress the importance of alternative splice variant mapping. If not performed, this may lead to false interpretations of the results.

Cell survival was measured by the MTS assay [81]. After HAL-PDT, 100 μl of each sample was placed to 96-well plastic microplates (Nunc) and incubated at 37°C for 0, 1 and 3 h, followed by the addition of 20 μl of MTS (5 mg/ml) (Promega Corporation, Madisom, WI, USA) into each well for an additional 1 h-incubation. The absorbance at 490 nm was measured with a microplate reader (Labsystems Oy, Helsinki, Finland). The absorbance of blank wells containing medium and MTS, but no cells, was subtracted from all readings and cell survival was expressed as the fraction of control samples.

Total RNA was isolated using GenElute Mammalian Total RNA kit (Sigma, St. Louis, MO) according to the manufacturer's protocol. RNA concentrations were determined with NanoDrop spectrophotometer (NanoDrop Technologies). The integrity and degree of degradation of RNAs was calculated using Agilent 2100 Bioanalyzer (Agilent Technologies AS). Fluorescence-labelled cDNAs were synthesized from 20 μg of the total RNA using an indirect amino allyl microarray labelling kit (Faiplay, Stratagene, La Jolla, CA, USA) according to the manufacturer's recommendations.

The cDNA microarrays used in this study were obtained from The Norwegian Microarray Consortium, for details on the arrays, we refer to [82]. The targets, approximately 15.000 unique I.M.A.G.E cDNA clones were from ResGen 40 k set. The hybridization volume of 110 μl consisted of: 8–10 μl of each of the labelled probes, 16 μg poly A (Amersham Biosciences), 15 μg human Cot-1 DNA (Invitrogen) and 85–90 μl of Microarray Hybridization Buffer #1 from Ambion (Austin, TX). The final mix was heated for 2 min at 100°C and after spinning down it was applied on a microarray. A hybridization station (Genomic Solutions, Inc., Ann Arbor, MI) was used for hybridization and wash, the details on hybridizations and wash can be found [82].

For protein isolation, the cell pellets were lysed on ice in 1 ml of lysis buffer (0.4% w/v SDS, 5 mM EDTA, 5 mM EGTA, 10 mM sodium pyrophosphate and 20 mM Tris-base; pH 7.2) for 30 min, briefly sonicated, diluted 1:2 in double strength SDS gel-loading buffer [double strength, 1% (w/v) SDS, 4.8 mM sodium deoxycholate, 10% (v/v) mercaptoethanol, 1% (v/v) Igepal CA-630, ~0.1% (w/v) Bromophenol Blue, 13,4% (v/v) glycerol and 120 mM Tris/HCl, pH 6.8] and boiled for 5 min at 95°C. After measuring the protein contents of the extracts with the BCA protein assay kit from Pierce (Rockford, USA) [92], 10 μg of samples were separated by SDS gel electrophoresis for 40 min at 200 V in 10% polyacrylamide gels containing 0.1% SDS. Molecular weight markers were included in all gels. Gel-separated proteins were transferred to nitrocellulose blotting membranes using a semi-dry transfer unit (Bio-Rad Laboratories, Hercules, CA, USA) with Towbin's blotting buffer (192 mM glycine, 20% methanol and 25 mM Tris-base; pH 8.3). The membranes were blocked by overnight incubation with 5% dry milk in TBS containing 0.2% Tween-20 (TBS-T) at 4°C, and washed three times for 10 min each in TBS-T. For detection of proteins, the membranes were incubated with the antibodies of MYC, ATF3 and JUN (diluted 1:1000 in TBS-T) overnight at 4°C, washed three times with TBS-T and incubated with the respective anti-rabbit or mouse-horseradish peroxidase-conjugated secondary antibodies (diluted 1:2000 in TBS-T) for 1 h at room temperature before being visualized by chemiluminescence using an ECL Western Detection Kit (Amersham Biosciences). To verify optimal blotting condition, the remaining proteins in the polyacrylamide gels were routinely stained with coomassie solution (0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol, and 10% (v/v) acetic acid) for 2 hours and excess staining was removed by using a destaining solution (50% (v/v) methanol in water with 10% (v/v) acetic acid) over night.

The same source of total RNAs (as in the microarray experiments) was used for real-time RT-PCR validation of selected genes. cDNA was synthesized from 1 μg DNase I-treated RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The cDNA solution was diluted 1:3 or 1:6 with nuclease-free water. Real-time PCR was performed using the iQ SYBR Green supermix (Bio-Rad) and specific primer pairs for the selected genes (specified in Fig. Fig.55 and Table 6). Primers were designed using the software Primer Express 2.0 (Applied Biosystems, Foster City, CA). For each sample the following mix (was prepared: 10 μl cDNA, 30 μl iQ SYBRGreen Supermix, 300 nM of each primer (MWG Biotech AG, Germany) and nuclease-free water to a final volume of 60 μl. Aliquots of 25 μl were distributed in two wells on the PCR plate. Real-time PCR reactions were run on an iCycler (Bio-Rad) with the following amplification protocol: 3 min initial denaturation at 95°C, 50 cycles of 10 s denaturation at 95°C and 35 s annealing/extension at 60°C. A final melt curve analysis was included to verify that one specific product was obtained in each reaction. The fold changes in the relative gene expression were calculated using the Gene Expression Macro, version 1.1 (Bio-Rad). Calculations were based on the ΔΔCt method, in which the threshold cycle number (Ct) for each studied gene in each sample were normalised to the Ct-value of the reference gene in the same sample. As a reference gene we used human acidic ribosomal phosphoprotein P0 (RPLPO) and TATA binding protein (TBP). Both refrence genes were tested and showed not to be affected by the photodynamic treatment. The normalized values (ΔCt) in the control sample were given value of 1, and the fold-change between the photodynamic treated samples and the controls were calculated accordingly.