In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells.

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.

Changing intestinal connective tissue interactions alters homeobox gene expression in epithelial cells.

In segmented organs, homeobox genes are involved in axial patterning and cell identity. Much less is known about their role in non-segmented endoderm derivatives such as the digestive epithelium. Using a xenograft model of fetal intestinal anlagen implanted under the skin of nude mice, we have investigated whether the expression of five homeobox genes (HoxA-4, HoxA-9, HoxC-8, Cdx-1 and Cdx-2) is modified when intestinal epithelium undergoes normal development or displays heterodifferentiation in association with heterotopic mesenchyme. In homotypic associations of fetal endoderm and mesenchyme that recapitulate normal development, the overall pattern of homeobox gene expression was maintained: HoxA-9 and HoxC-8 were the highest in the colon and ileum, respectively, and HoxA-4 was expressed all along the intestine; Cdx-1 and Cdx-2 exhibited an increasing gradient of expression from small intestine to colon. Yet, grafting per se caused a faint upregulation of HoxA-9 and HoxC-8 in small intestinal regions in which these genes are not normally expressed, while the endoderm-mesenchyme dissociation-association step provoked a decay of Cdx-1 in the colon. In heterotopic associations of colonic endoderm with small intestinal mesenchyme, the colonic epithelium exhibited heterodifferentiation to a small intestinal-like phenotype. In this case, we observed a decay of HoxA-9 expression and an upregulation of HoxC-8. Additionally, heterodifferentiation of the colonic epithelium was accompanied by a downregulation of Cdx-1 and Cdx-2 to a level similar to that found in the normal small intestine. To demonstrate that mesenchyme-derived cells can influence Cdx-1 and Cdx-2 expression in the bowel epithelium, fetal jejunal endoderm was associated with intestinal fibroblastic cell lines that either support small intestinal-like or colonic-like morphogenesis. A lower expression of both homeobox genes was shown in grafts presenting the small intestinal phenotype than in those showing glandular colonic-like differentiation. Taken together, these results suggest that homeobox genes participate in the control of the positional information and/or cell differentiation in the intestinal epithelium. They also indicate that the level of Cdx-1 and Cdx-2 homeobox gene expression is influenced by epithelial-mesenchymal cell interactions in the intestinal mucosa.

Mesenchyme-mediated effects of retinoic acid during rat intestinal development.

In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endodermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as alpha1 and beta1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mesenchymal-epithelial cell interactions during intestinal development.

Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development.

In rodents, the intestinal tract progressively acquires a functional regionalization during postnatal development. Using lactase-phlorizin hydrolase as a marker, we have analyzed in a xenograft model the ontogenic potencies of fetal rat intestinal segments taken prior to endoderm cytodifferentiation. Segments from the presumptive proximal jejunum and distal ileum grafted in nude mice developed correct spatial and temporal patterns of lactase protein and mRNA expression, which reproduced the normal pre- and post-weaning conditions. Segments from the fetal colon showed a faint lactase immunostaining 8-10 d after transplantation in chick embryos but not in mice; it is consistent with the transient expression of this enzyme in the colon of rat neonates. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and distal ileum developed as xenografts in nude mice, and they exhibited lactase mRNA and protein expression patterns that were typical of the origin of the endodermal moiety. Endoderm from the distal ileum also expressed a normal lactase pattern when it was associated to fetal skin fibroblasts, while the fibroblasts differentiated into muscle layers containing alpha-smooth-muscle actin. Noteworthy, associations comprising colon endoderm and small intestinal mesenchyme showed a typical small intestinal morphology and expressed the digestive enzyme sucrase-isomaltase normally absent in the colon. However, in heterologous associations comprising lung or stomach endoderm and small intestinal mesenchyme, the epithelial compartment expressed markers in accordance to their tissue of origin but neither intestinal lactase nor sucrase-isomaltase. A thick intestinal muscle coat in which cells expressed alpha-smooth-muscle actin surrounded the grafts. The results demonstrate that: (a) the temporal and positional information needed for intestinal ontogeny up to the post-weaning stage results from an intrinsic program that is fixed in mammalian fetuses prior to endoderm cytodifferentiation; (b) this temporal and positional information is primarily carried by the endodermal moiety which is also able to change the fate of heterologous mesodermal cells to form intestinal mesenchyme; and (c) the small intestinal mesenchyme in turn may deliver instructive information as shown in association with colonic endoderm; yet this effect is not obvious with nonintestinal endoderms.

Adhesive properties and integrin expression profiles of two colonic cancer populations differing by their spreading on laminin.

The mostly undifferentiated parental HT29 (HT29p) human colonic adenocarcinoma cell line and a differentiated subpopulation selected by the anti-cancer drug 5-fluorouracil (HT29-Fu) (Lesuffleur et al. (1991) Int. J. Cancer 49, 721-730) display strikingly different behavior when grown on laminin coatings: the former grows as aggregates while the latter grows as monolayers. In an attempt to explain this difference, we performed a comparative study of cell adhesion properties and of expression, involvement and localization of the alpha 6, beta 1 and beta 4 subunits constituting the integrin family among the two cell populations. HT29p and HT29-Fu cells exhibited a similar adhesion pattern to laminin and laminin fragments E8 and P1. In both cell lines, cell adhesion could be blocked at about 90% with anti-alpha 6 subunit antibodies and around 30-50% with anti-beta 1 antibodies; no inhibition of the cell adhesion was obvious when using anti-beta 4 antibodies. Immunoprecipitations of iodinated membrane-solubilized proteins and immunoblotting experiments showed that all alpha 6 chains expressed in both HT29p and HT29-Fu cell populations exist as alpha 6 beta 4 integrins; beta 1 subunits are associated with alpha 2 and alpha 3 chains. When HT29p or HT29-Fu cells were injected subcutaneously in nude mice, a similar expression pattern of alpha 6, beta 4 and beta 1 integrin subunits was noticeable in the resulting tumors: alpha 6 and beta 4 subunits were localized at the basal surface of the tumor cells facing the stromal elements, and to a lesser extent at the cell-cell contacts within the tumor-cell clumps; beta 1 subunits were mainly found within the cytoplasm of the tumor cells. Despite these overall similarities among the two cell lines, the following changes could account for their different behavior on laminin: less proteolytic processing of the beta 4 integrin subunit occurred in HT29-Fu cells yielding peptidic fragments of 175 kDa, which are absent from the parental cells; the immunostaining pattern of the various subunits demonstrated a segregation of alpha 6, beta 4 and beta 1 integrin subunits on the basal side of the HT29-Fu cells when cultured on laminin to the detriment of their lateral location, a phenomenon that was not obvious in the parental cells. Altogether, these results suggest that the distinct behavior of the undifferentiated versus differentiated HT29 cell populations on laminin is not related to altered adhesion properties of the cells but rather to a deficient stabilization of the adhesion leading to cell spreading.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental photodynamic therapy with a copper metal vapor laser in colorectal cancer.

In an attempt to define the best conditions for an adjunctive treatment of residual colonic microtumors by photodynamic therapy (PDT), an experimental model has been defined. S.c. HT29 colonic-cancer-cell tumors grown in nude mice were used and, 48 hr after i.p. administration of 30 mg/kg Photofrin (PH), laser illumination was performed with 75 or 150 Joules/cm2. The efficiencies of 2 lasers, the classically used rhodamine laser (RL) and a copper metal vapor laser (CMVL), were compared. The effects of PDT were assessed by histological and immunocytochemical (detection of a digestive enzyme, dipeptidyl-peptidase IV, as a marker of cell viability) follow-up and by the growth curve of the tumors after illumination. We conclude that, although the depth of necrosis resulting from PDT was nearly 3 mm at 75 J/cm2 and nearly 4-5 mm at 150 J/cm2 with both lasers, complete necrosis was obtained only with the CMVL at 150 J/cm2 (in 50% of the tumors). Under the other conditions, a layer of unaffected cells persisted at the pole opposite to laser illumination, resulting in growth curves lower than but parallel to those of the controls. Analysis of drug concentrations in the tumors and various organs, 48 hr after injection, i.e., at the time of laser illumination, revealed the presence of 21 micrograms/g dry weight PH in the tumors. The tumor vs. host-organ ratios were equal to or higher than 1 for the small bowel, colon, stomach, lung, skin and muscle. In contrast, the ratios were below 1 for the spleen, pancreas, kidney and liver.

Changes in the expression of laminin during intestinal development.

The expression of laminin, a major glycoprotein constituent of basement membranes, was investigated in the rat developing intestine. The biosynthesis of laminin was studied after metabolic labeling of intestinal segments taken at various stages of development; the neosynthesized laminin was purified by affinity chromatography on heparin-Sepharose. Immunoblotting and immunoprecipitation experiments allowed us to analyze its constitutive chains. The data show that laminin is synthesized in very large amounts at 16-18 days of gestation concomitant with the onset of intestinal morphogenetic movements, i.e. villus emergence. Evaluation of the relative proportion of individual laminin polypeptides shows that laminin B1/B2 chains are produced in excess of A chains whatever the developmental stage considered. Interestingly at 17 days of gestation, levels of laminin A subunits are maximal. A second rise in the A/B chain ratio starts around birth and continues until adulthood. These quantitative data are corroborated by the immunocytochemical detection of laminin A and B chains, which revealed a specific spatiotemporal pattern. The finding that laminin A chains are located in the basement membrane of growing villi and of adult crypts raises the possibility that they may be involved in the process of cell growth and/or in the establishment of cell polarity by creating a specialized extracellular microenvironment.

Changes in glycosaminoglycan synthesis and in heparan sulfate deposition in human colorectal adenocarcinomas.

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures. Fibroblastic cell-mediated effects of glucocorticoids.

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.