Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice.

Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.

Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein.

Targeted gene delivery to vascular lesions is a major challenge in the development of gene therapy protocols for cardiovascular diseases. One approach would be to enable retroviral vectors to accumulate at sites of vascular injury and enhance local vector concentration. An early step in wound repair is the adhesion of platelets to collagen exposed from damaged vasculature. Hence, the Moloney murine leukemia virus (MoMLV) envelope (env) protein was engineered to incorporate a high-affinity collagen-binding domain derived from von Willebrand clotting factor, and expressed in Escherichia coli and in mammalian cells. The chimeric env protein bound tightly to collagen, and virions bearing this collagen-binding env protein exhibited viral titers approaching those of virions expressing wild-type (WT) env protein. The chimeric virions were concentrated on collagen matrices, and they retained their infectivity under conditions in which virions bearing WT env protein were washed away. Targeted delivery of the chimeric env protein to injured mouse aorta and selective binding of the collagen-targeted virions to injured rabbit artery were observed. In comparative studies, vascular smooth muscle cell transduction was demonstrated in catheter-injured carotid arteries following infusion of collagen-targeted virions but not of virions bearing WT env protein. Taken together, these observations demonstrate the ability of collagen-targeted virions to localize gene delivery to sites of vascular injury.

Early regional expression and secretion of peptide YY and enteroglucagon after massive resection of small bowel.

BACKGROUND: Previous studies suggest that peptide YY (PYY) and enteroglucagon have an important role in intestinal adaptation after massive small bowel resection. This study was done to define the mechanisms, timing, and anatomic distribution of the PYY and enteroglucagon response. STUDY DESIGN: Lewis rats underwent resection of 70 percent of the small bowel (leaving equal segments of jejunum and ileum), transection, or laparotomy alone. Jejunum, ileum, and colon were compared in resected, transected, and control bowel six hours, 24 hours, one week, and two weeks postoperatively. RESULTS: Analysis of DNA, RNA, and protein per cm of bowel demonstrated hyperplastic changes. Radioimmunoassay revealed plasma PYY and enteroglucagon to be significantly elevated 24 hours after resection and they remained so through week two. In contrast, tissue PYY and enteroglucagon content decreased significantly in all tissues (p < 0.05) after resection. Reverse transcriptase polymerase chain reaction and Southern blot analysis demonstrated an immediate and sustained increase in PYY messenger RNA (mRNA) in both the ileum (fourfold) and in the colon (2.5-fold) at six hours (p < 0.05). A gradual increase in PYY mRNA was also demonstrated in the jejunum with significance at two weeks (p < 0.05). Proglucagon mRNA was significantly higher in the jejunum, compared with the ileum and colon, at 24 hours, one week, and two weeks postresection. CONCLUSIONS: Alterations in PYY and enteroglucagon synthesis occur early in the ileum and colon after massive small bowel resection. The residual jejunum, however, is primarily responsible for the adaptive hyperenteroglucagonemia. These findings suggest that although PYY and enteroglucagon are colocalized to the same cell type, there is a gene-specific response for these two peptides after resection.

Pancreatic peptide YY mRNA levels increase during adaptation after small intestinal resection.

Peptide YY (PYY) is a 36-amino-acid peptide known to inhibit pancreatic and gastrointestinal secretion. Immediately following small bowel resection, intestinal PYY mRNA and plasma PYY levels rise. The purpose of this study was to determine whether PYY expression changes in the pancreas during the adaptive period after extensive small bowel resection. Female Sprague-Dawley rats (250 g) underwent 70% small intestinal resection or transection alone as control. Animals were sacrificed at 6 hr, 24 hr, 1 week, or 2 weeks following operation (N = 5/time group). Pancreatic tissue was harvested and RNA was isolated by the guanididium-thiocyanate method. PYY mRNA was analyzed by reverse transcriptase PCR, standardized to glyceraldehyde-3-phosphate dehydrogenase, and semiquantitated by Southern blotting and 32P cpm. Ribonuclease protection assay was used to confirm PCR results. PYY mRNA expression was increased 9 1/2-fold beginning 6 hr after resection compared to transection (P < 0.05). PYY mRNA levels remain elevated, 2 1/4-fold greater than control after 2 weeks (P < 0.05) as analyzed by reverse transcriptase PCR and ribonuclease protection assay. Quantitation by ribonuclease protection assay reveals a gradual elevation of PYY mRNA levels in transected animals compared to a nonoperated rat starting at 1 and 2 weeks. Pancreatic PYY mRNA levels increase rapidly after extensive intestinal resection and remain elevated 2 weeks postoperatively. These results confirm for the first time that the increase in PYY seen after extensive intestinal resection also occurs in extraintestinal sites. In the pancreas, elevated PYY levels may inhibit exocrine secretion, reducing luminal volume, and thereby facilitating intestinal adaptation.

Up-regulation of Na+,K+ adenosine triphosphatase after massive intestinal resection.

BACKGROUND: The mechanisms of intestinal adaptation after resection are not completely defined. The purpose of this study was to examine the changes after resection in the enterocyte basolateral Na+,K+ adenosine triphosphatase (ATPase) known to play a critical role in epithelial transport and homeostasis. METHODS: Lewis rats underwent 70% small bowel resection or transection. At 6 hours, 24 hours, 1 week, and 2 weeks, jejunum and ileum were harvested for analysis of Na+,K+ ATPase activity, kinetic analysis, and alpha 1-ATPase messenger RNA and protein levels. RESULTS: Na+,K+ ATPase activity increased (p < 0.05) in both the jejunum and ileum by 2 weeks after resection. This rise in activity correlated with an increase in the maximal activity of ATPase, from 20.8 to 101.01 mumol inorganic phosphate.mg-1.hr-1. ATPase messenger RNA levels increased sixfold in the jejunum and tenfold in the ileum by 2 weeks after resection (p < 0.05). Protein levels rose at 6 hours and remained elevated in both tissues. CONCLUSIONS: After intestinal resection, enterocyte Na+,K+ ATPase activity rises as a result of an increase in the number of transporters per cell. This occurs through both transcriptional and translational mechanisms. It appears that intestinal adaptation after resection involves not only an increase in absorptive surface area but also functional adaptation by the individual enterocyte.

Adaptation of the Na+/glucose cotransporter following intestinal resection.

Following massive small bowel resection, the remaining intestine adapts to compensate for lost absorptive capacity. Although the Na+/glucose cotransporter plays a critical role in nutrient, fluid, and electrolyte transport in the small intestine, its role in adaptation following resection has not been defined. To examine this, we sought to determine whether there were changes in the expression of the Na+/glucose cotransporter, SGTL1, at the messenger RNA level. Lewis rats underwent either transection or 70% small bowel resection and reanastomosis. The animals were sacrificed at intervals following operation. Jejunum proximal to the anastomosis and ileum and colon distal to the anastomosis were harvested and analyzed for Na+/glucose mRNA by reverse transcriptase-polymerase chain reaction and Southern blot. Blots were semiquantitated by 32P labeling and standardized to beta-actin. Histologic sections and analysis of DNA, RNA, and protein content revealed hyperplastic changes. Following resection, mRNA for the Na+/glucose cotransporter in the jejunum increased significantly (P < 0.05) by 1 week and remained elevated. In the ileum, an almost fivefold increase occurred at 6 hr and persisted throughout the study (P < 0.05). The early response was greater in the ileum, distal to the reanastomosis, than that in the jejunum (P < 0.05). In contrast, there was no change in the small amount of transporter mRNA detected in the colon. These results suggest that, in addition to mucosal hyperplasia, the intestinal response to resection involves upregulation of transporter mRNA by the individual enterocyte. This transcriptional increase in the Na+/glucose cotransporter appears to be an early response by the intestine and may be important in maintaining overall intestinal transport capacity following resection.

Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.