Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects.

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL(DR5), had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL(DR4) when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.

Sunburn reaction in the dorsal skin of hypotrichotic WBN/ILA-Ht rats.

The dorsal skin responses to a single irradiation with a high-dose of UVB (10kJ/m2) were examined histologically and immunohistochemically in UVB-sensitive Wistar-derived hypotrichotic WBN/ILA-Ht rats (HtRs). Sunburn cells (SBCs) which were characterized by pyknotic nuclei and eosinophilic cytoplasm and had ultrastructual characteristics of apoptotic cells were first observed in the epidermis at 3 hours (h) after irradiation. The number peaked at 6 h, and then decreased rapidly. The expressions of p53 protein, which is known to be closely related to the formation of SBCs, and of p21 protein, which is one of the transcriptional target genes of p53, were immunohistochemically detected, and their labeling index (LI) in the epidermis peaked at 12 to 24 h (p53) or at 24h (p21) after irradiation. On the other hand, proliferating cell nuclear antigen (PCNA)-LI in keratinocytes was significantly lower than the control group at 6 h after irradiation and thereafter it increased and became significantly higher than the control group from 24 to 48 h. At 48 h, moderate hyperplasia with moderate numbers of mitotic keratinocytes was first observed in the epidermis. In the dermis, mild edema developed from 12 to 36 h and it accompanied mild lymphocyte infiltration at 36 h. Judging from the present results, it was suggested that some factors other than p53 might be involved in SBC formation, and that p53 might induce p21 protein and play an important role in cell growth arrest in keratinocytes after UVB irradiation.

Kinetics of cytokines mRnas expression in the dorsal skin of WBN/ILA-Ht rats following topical application of T-2 toxin.

The kinetics of cytokines mRNAs expression was examined in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats topically applied with T-2 toxin. After the application of 10 microl (0.5 microg/microl) of T-2 toxin solution, the total mRNA was obtained from skin biopsies at 3, 6, 12 and 24 hours after treatment (HAT). Reverse transcription-polymerase chain reaction (RT-PCR) was carried out with pairs of oligonucleotide primers corresponding to the cDNA sequences of rat TNF-alpha, IL-1 alpha, IL-1beta, IL-6 and IL-10 cytokines. The level of TNF-alpha mRNA showed marked elevation at 3HAT and decreased toward 24HAT, but it remained significantly higher level even at 24HAT. In addition, the level of IL- 1beta mRNA expression showed a sligth but significant elevation at 3 and 24HAT. On the other hand, no significant differences were observed in other cytokines mRNAs expression between T-2 toxin-treated and control groups througth the observation period. Together with our previous report describing the sequence of epidermal cell apoptosis (Albarenque et al. 1999), the present results suggest that the elevation of TNF-alpha mRNA expression may play an important role in T-2 toxin-induced epidermal cell apoptosis.

Kinetics of apoptosis-related genes mRNA expression in the dorsal skin of hypotrichotic WBN/ILA-ht rats after topical application of T-2 toxin.

The expression of apoptosis-related genes mRNAs was examined in the dorsal skin of hypotrichotic WBN/ILA-Ht rats topically applied with T-2 toxin (10 microl of 0.5 microg/microl solution). The total mRNA was obtained from skin biopsy samples from each rat at 3, 6, 12 and 24 hours after T-2 toxin treatment (HAT), and RT-PCR was carried out with pairs of oligonucleotide primers corresponding to the cDNA sequences of rat p53, bcl-2, c-ki-ras, c-fos and c-jun oncogenes. The expression of c-fos mRNA markedly increased at 3 HAT, peaked at 6 HAT, and greatly decreased at 12 HAT. However it maintained a higher level, compared with the control level, even at 24 HAT. Although not prominent, the expression of c-jun mRNA also showed significant elevation from 3 to 12 HAT. On the other hand, there were no changes in the expression of p53, bcl-2 and c-ki-ras mRNAs throughout the observation period. Judging from the present results and our previous report that epidermal cells developed apoptosis at 12 HAT (Histol Histopathol 1999; 14: 337-342), the induction of c-fos and perhaps of c-jun mRNAs may be associated with T-2 toxin-induced epidermal cell apoptosis.

Kinetics and distribution of transforming growth factor (TGF)-beta 1 mRNA in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following topical application of T-2 toxin.

Depression of basal cell proliferating activity and subsequent induction of basal cell apoptosis in the epidermis and infiltration of inflammatory cells including mast cells in the dermis were observed in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following the topical application of T-2 toxin in our previous study (ALBARENQUE et al. 1999). In the present study, kinetics of TGF-beta 1 mRNA was investigated using the same experimental system. The level of TGF-beta 1 mRNA of the whole skin tissue measured by competitive RT-PCR method showed a slight elevation from 6 to 12 hours after treatment (HAT) and reached the significantly higher level at 24HAT compared with the control skin. The increase in signals of TGF-beta 1 mRNA detected by in situ hybridization method started at 3HAT in the epidermis and progressed thereafter both in the epidermis and in the dermis. These results suggest that the elevated level of TGF-beta 1 mRNA may have a close relation to the induction of epidermal basal cell apoptosis as well as to the intradermal infiltration of mast cells and fibroblasts following the topical application of T-2 toxin.

T-2 toxin-induced acute skin lesions in Wistar-derived hypotrichotic WBN/ILA-Ht rats.

Acute lesions in the dorsal skin topically applied with T-2 toxin (10 microliters of 0.5 mg/ml-solution to 1 cm2) were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats up to 24 hours after treatment (24HAT). In the epidermis, depression of basal cell proliferating activity was detected at 3HAT by immunostaining for proliferating cell nuclear antigen (PCNA), and the percentage of PCNA-positive basal cells decreased thereafter. At 12HAT, in addition to intracytoplasmic edema of spinous cells, acidophilic degeneration of basal cells characterized by shrinkage of cell body with acidophilic cytoplasm and pyknotic or karyorrhectic nuclei became prominent. Most of these nuclei were positive for TUNEL which is a widely used immunostaining for the in situ detection of fragmented DNA, i.e. apoptosis, and the percentage of TUNEL-positive basal cells increased thereafter. The nuclei of these basal cells also showed ultrastructural changes characteristic for apoptosis. On the other hand, in the dermis, infiltration of inflammatory cells including mast cells started at 3HAT and increased thereafter. In addition, capillary and small vessel endothelial degeneration developed at 6HAT and progressed thereafter. These results suggest that T-2 toxin directly affects the epidermis and produces apoptosis in basal cells.