Decreased efficiency of trypsinization of cells following photodynamic therapy: Evaluation of a role for tissue transglutaminase

Photochemistry and Photobiology, Jan 2001 by Ball, Denise J, Mayhew, Stephen, Vernon, David I, Griffin, Martin, Brown, Stanley B

Decreased Efficiency of Trypsinization of Cells Following Photodynamic Therapy: Evaluation of a Role for Tissue Transglutaminase*

ABSTRACT

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm-2 (3 J cm CM-2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm-2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorin (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPCPDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

tAbbreviations: DMEM, Dulbecco's minimal essential medium; ECM, extracellular matrix; EDTA, ethylene diamine tetraacetic acid; FCS, fetal calf serum; HpD, haematoporphyrin derivative;

LD50, extracellular concentration resulting in 50% MTT reduction; m-THPC, 5,10,15,20 meso-tetra(hydroxyphenyl)chlorin; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PDT, photodynamic therapy; PHP, polyhaematoporphyrin; PPC, pyridinium zinc (II) phthalocyanine; TBS, tris buffered saline; tTGase, tissue transglutaminase.

INTRODUCTION

The response of cells to photodynamic therapy (PDT)t appears to depend on a number of factors, one of which is the sensitizer used. Following the development of Photofrin , numerous so-called second generation sensitizers have been developed which have shown good activity in vitro and in vivo (1-3). Charge and hydrophobicity appear to be the main factors governing cellular localization (1), with localization being the major determinant of cell death (2,4). Membrane damage has been reported following photosensitization with hematoporphyrin derivative (HpD) (5). This has been suggested to affect cancer cell adhesiveness (6), which may subsequently have consequences on metastasis (7).

The field of cellular adhesion is a vast and expanding area of research. Cells attach to the substratum via adhesion plaques, or focal adhesions (8). Originally thought to serve as a site of structural linkage, there is now compelling evidence that this interaction can mediate a number of biochemical events including cell division, cell differentiation and apoptosis (9-11).

Tissue transglutaminase (tTGase, type 2, EC 2.3.1.13) belongs to a family of enzymes that catalyze the posttranslational modification of proteins via an acyl transfer reaction between peptide-bound glutamine residues and primary amines, resulting in the formation of covalent E-(y-glutamyl)lysine crosslinks in proteins (12). Plasma transglutaminase (factor XIII), which carries out stabilization of the fibrin clot during coagulation, is probably the most wellcharacterized member of this family (12).

The biological function of tTGase is not completely understood. It is a calcium-dependent GTP binding protein, that is found in nearly all tissues (13-15). It has been implicated in a number of cellular events including increasing adhesivity (16), assembly and stabilization of the extracellular matrix (ECM) (17), induction of morphological changes (16), apoptotic body formation (18-20), differentiation (21) and also as a GTP binding protein (14).

In vitro, the increase of tTGase is often paralleled by an increased attachment to the substratum and a concomitant resistance to detachment following incubation with trypsin (17). Although an increased resistance to trypsinization following PDT has been reported (6,22), evidence for the mechanisms) of this has been slow to emerge. In this report, we have used three previously characterized photosensitizers (2), pyridinium zinc (II) phthalocyanine (PPC), 5,10,15,20 mesa-tetra(hydroxyphenyl) chlorin (m-THPC) and polyhaematoporphyrin (PHP) (23), a compound identical to Photofrin, to investigate cell adhesion following PDT in three cell lines with different basal levels of tTGase activity. It has also been proposed that tTGase activation may cause a form of cell death, distinct from apoptosis and necrosis, in which extensive crosslinking in the cytoplasm and cell-substratum contacts preserves both cell integrity and adhesion to the ECM (24). Therefore the PDT response of metastatic hamster fibrosarcoma cells (Met B) transfected with human tTGase has been compared with that of control Met B cells, in an attempt to investigate the role, if any, of tTGase following PDT-mediated stress.