Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

Direct serum injection high-performance liquid chromatographic method for the simultaneous determination of phenobarbital, carbamazepine and phenytoin

A method is described for the simultaneous measurement of serum levels of three antiepileptic drugs, phenobarbital, phenytoin and carbamazepine, by direct injection high-performance liquid chromatography on a 25-cm Pinkerton internal surface reversed-phase (ISRP) column. Several commonly available compounds were tested and found not to co-chromatograph with the three drugs of interest or the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin. Results obtained on patients' samples with this method compared well with those from enzyme-multiplied immunoassay technique (EMIT).     

Breakup of 8B and the 7Be(p, γ)8B reaction

The calculated rate of events in some of the existing solar neutrino detectors is directly proportional to the rate of the ⁷Be(p, γ)⁸B reaction measured in the laboratory at low energies. However, the low-energy cross sections of this reaction are quite uncertain as various measurements differ from each other by 30–40%. The Coulomb dissociation process which reverses the radiative capture by the dissociation of ⁸B in the Coulomb field of a target, provides an alternate way of accessing this reaction. While this method has several advantages (like large breakup cross sections and flexibility in the kinematics), the difficulties arise from the possible interference by the nuclear interactions, uncertainties in the contributions of the various multipoles and the higher order effects, which should be considered carefully. We review the progress made so far in the experimental measurements and theoretical analysis of the breakup of ⁸B and discuss the current status of the low-energy cross sections (or the astrophysical S-factor) of the ⁷Be(p, γ)⁸B reaction extracted therefrom. The future directions of the experimental and theoretical investigations are also suggested. Work supported by EPSRC, UK, grant nos J/95867 and L/94574.     

Photodynamic therapy resistant human colon carcinoma HT29 cells show cross-resistance to UVA but not UVC light

The isolation of photodynamic therapy (PDT)-resistant HT29 human colon adenocarcinoma cells has been reported previously. These PDT-resistant variants show increased expression of the Hsp27 and BNip3 proteins and a decreased expression of mutant p53 protein compared with parental HT29 cells. Because mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. In the present report, we examined the colony survival of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells to ultraviolet light (UV) treatment. The HT29 PDT-resistant variants showed cross-resistance to long-wavelength UVA (320-400 nm) but not to short-wavelength UVC (200-280 nm) light. Cell sensitivity to UVA or UVC was then correlated with Hsp27, BNip3 and mutant p53 protein levels in the PDT-resistant variants as well as in several clonal variants of HT29 cells that express different levels of Hsp27, BNip3 and mutant p53. We show that increased expression of Hsp27 and BNip3 and decreased expression of mutant p53 correlated with increased resistance to UVA. In contrast, increased expression of Hsp27 and BNip3 correlated with increased sensitivity to UVC, whereas increased expression of mutant p53 showed no significant correlation with sensitivity to UVC. These results suggest that the PDT-resistant HT29 cell variants are differentially sensitized to UVA compared with UVC due, in part at least, through the altered expression levels of BNip3, Hsp27 and mutant p53.     

Up-regulation of Hsp27 plays a role in the resistance of human colon carcinoma HT29 cells to photooxidative stress

The Photofrin-resistant cell line (HT29-P14) was used in the present study to investigate the mechanism(s) involved in Photofrin-mediated photodynamic therapy (PDT). We compared gene expression profiles between the resistant cell line and its parental cell line (HT29) using DNA microarray analysis. A significant up-regulation of small heat shock protein 27 (Hsp27) was found in HT29-P14 cells. The elevated Hsp27 level may play an important role in the resistance of HT29-P14 to Photofrin-PDT. To test this hypothesis, we stably transfected HT29 cells with human Hsp27 complementary DNA. The potential role of Hsp27 in the resistance to PDT was examined in Hsp27-overexpressing cells. Stable trasnfected cells (H13) showed an increased survival after Photofrin-PDT, suggesting that the up-regulation of Hsp27 is related to the induced resistance to Photofrin-PDT. Phosphorylation of Hsp27 has been suggested to play an important role in cytoprotection. We have examined the phosphorylation activity of Hsp27 among the parental and resistant cells, as well as the overexpression cells. An elevated level of Hsp27 resulted in an increased ability of phosphorylation in both resistant and overexpressing cells after PDT. The activation of the phosphorylation of Hsp27 induced by PDT was not mediated by the p38 mitogen-activated protein kinase. These data suggest that Hsp27 may play an important role in mediating the adaptive response to Photofrin-PDT-induced oxidative stress and that the pathways leading to Hsp27 phosphorylation may contribute to the resistance of the cells to photooxidative damage.     

In vivo resistance to photofrin-mediated photodynamic therapy in radiation-induced fibrosarcoma cells resistant to in vitro Photofrin-mediated photodynamic therapy.

The effects of Photofrin-mediated photodynamic therapy (PDT) on the in vitro cell survival and in vivo tumor growth of murine radiation-induced fibrosarcoma (RIF) cell tumors have been examined following in vivo PDT treatment of tumors. The response to in vivo PDT is examined in tumors derived from RIF-1 mouse fibrosarcoma cells and in tumors derived from RIF-8A cells, which show in vitro resistance to PDT. A significant reduction in tumor volume is observed over the first three days following in vivo PDT treatment of either 5 or 10 mg/ kg. The reduction in tumor volume is greater for a 10 compared to a 5 mg/ml dose and occurs to a similar extent for both RIF-1 and RIF-8A tumors. The re-growth is significantly delayed for RIF-1 compared to RIF-8A tumors, indicating a greater response for RIF-1 tumors compared to RIF-8A tumors following PDT. A reduced response of the RIF-8A compared to the RIF-1 tumor cells is also observed in the clonogenic survival of cells from tumors that were excised and explanted in vitro immediately following in vivo PDT treatment. These data indicate that the intrinsic cell sensitivity to PDT is an important component in the mechanism that leads to tumor response following in vivo photodynamic therapy.     

Extreme dark cytotoxicity of Nile Blue A in normal human fibroblasts.

Early reports using mouse models indicated that Nile Blue A (NBA) is taken up more efficiently by tumor cells than normal tissue and retards tumor growth. NBA also shows both dark toxicity and phototoxicity of human tumor cells in vitro. However, studies on the dark toxicity of NBA and the effects of NBA-mediated photodynamic treatment in normal human cells are lacking. In the current study we have examined the cytotoxicity of NBA in normal human fibroblasts, spontaneously immortalized Li-Fraumeni Syndrome (LFS) cells and three different human tumor cell lines. The normal human fibroblasts showed extreme sensitivity to NBA compared with LFS cells and the human tumor cell lines. Treatment with 0.1 microgram/mL of NBA for 1 h reduced the colony formation of normal human fibroblasts by greater than 95%, but had no significant effect on the colony formation of LFS cells. No significant numbers of apoptotic cells were detected in either normal human fibroblasts or LFS cells following this drug concentration. Thus, unlike photodynamic therapy with some other photosensitizers, the dark toxicity of NBA was not caused by apoptosis. Although the drug uptake was higher in normal human fibroblasts compared with LFS cells, the difference in sensitivity between normal human fibroblasts and LFS cells could not be accounted for by the difference in drug uptake alone. In addition, we could not detect any significant photocytotoxic effect of NBA in either normal human fibroblasts or LFS cells for a drug concentration of 0.05 microgram/mL at light exposures of up to 6.7 J/cm2. These data indicate an extreme sensitivity of normal human fibroblasts to NBA and an inability to produce a significant photocytotoxic effect on human cells using NBA concentrations that have relatively low toxicity for normal human fibroblasts.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.