Significant growth inhibition and induction of apoptosis by IFN-beta in cancer cells including colorectal cancer cells have been observed. We and others have previously reported the Stat 1 induction of TRAIL is a crucial step in the IFN-beta induced apoptosis pathway. However, when evaluating the sensitivity of a panel of colorectal cancer cell lines, we found no clear correlation between activation of the Jak/Stat signaling pathway and response to interferon. In the present study, we have evaluated the interaction of the PI3k/Akt pathway and IFN-beta induced apoptosis in human colorectal cancer cells. The results demonstrate a correlation between Akt activity, phosphorylation of Bad and resistance to interferon-induced apoptosis in these cells. The association of activation of Akt, phosphorylation of Bad and resistance to IFN-beta-induced apoptosis was further supported by the observation that disruption of the pathway in a more resistant cell line led to sensitization, and expression of an activated Akt in a more sensitive cell line led to increased resistance. Taken together, this data indicates that the PI3/Akt kinase pathway may be an important contributor to IFN-beta sensitivity and resistance in colorectal cancer cells. This data demonstrates a potential pathway by which cells may develop resistance to IFN, and further elucidation of this process may allow us to better target IFN therapy.

Preclinical in vitro and in vivo studies have demonstrated synergistic interactions between 5-fluorouracil (5-FU) and type I and II IFNs against human colorectal cancer cells. Despite these activities, randomized human trials have failed to identify a clinical benefit for this combination treatment. These limited clinical results may be secondary to the short half-life of recombinant IFN protein and the increased systemic toxicities of 5-FU/IFN combinations. We have previously reported an adenoviral-mediated IFN-β gene therapy strategy, which may circumvent the pitfalls of recombinant IFN therapy. However, a dose-dependent toxicity and acute inflammatory response to systemically administered adenovirus vectors may limit the clinical application of this therapy. The combination of adenoviral-mediated IFN-β gene therapy and 5-FU resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model. These therapeutic effects were observed at a significantly lower vector dose than we had previously reported and with limited toxicity. This approach may allow for an effective clinical application of this therapy and warrants additional investigation.

Type I IFNs are known to inhibit tumor cell growth and stimulate the immune system. However, little is known of the mechanism of type I IFN-induced apoptosis in human cancer cells. In this study, we have IFN-beta treatment of a human colorectal cell line (KM12L4) and a resistant clone of this cell line, L4RIFN. We demonstrate the induction of apoptosis in the parent cell line. This process was associated with the induction of the Jak-Stat signaling pathway, induction of the proapoptotic mediator tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activation of procaspase-3, -8, -9, and -10. Additionally, we evaluated the role of Stat1 in mediating IFN-beta induction of these proapoptotic signals in a fibrosarcoma cell line (2ftgh) and a Stat1-deficient clone (U3A). Our results demonstrate that IFN-beta induction of apoptosis and the induction of proapoptotic mediator TRAIL is Stat1 dependent. Evaluation of a stable transfectant of the KM12L4 cell line expressing c-FLIP supports the role of TRAIL and the cell-surface death signaling pathways in IFN-beta induction of apoptosis. Studies evaluating the TRAIL promoter indicate induction of TRAIL promoter activity by IFN-beta. These results may represent a novel pathway by which IFN-beta may induce therapeutic effects.

Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-beta (hIFN-beta) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-beta in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-beta gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-beta (mIFN-beta) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-beta in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.

The membranes surrounding the chick embryo undergo striking morphological changes before hatching, which include structural degradation of the allantoic membrane. The fibrillar collagen content of the membranes declined by embryonic day (ED) 20 (the day of hatching). By ED 19, a 55-kDa matrix metalloproteinase (MMP) activity appeared in the extraembryonic fluid, and by ED 20 there was substantial 55-kDa MMP activity in embryonic membrane extracts. Reverse transcription-polymerase chain reaction was employed to clone a partial cDNA representing the chicken homologue of MMP-13, a 55- to 57-kDa enzyme. MMP-13 mRNA dramatically increased in abundance in embryonic membranes by ED 19, reaching a peak on ED 20. Introduction of the MMP inhibitor batimastat into the extraembryonic fluid prevented the structural changes in the embryonic membranes before hatching. We conclude that, like mammalian fetal membranes, chick embryonic membranes undergo terminal remodeling before hatching, in part as a result of increased MMP activity. The chicken egg system represents a novel in vivo model for exploring biochemical events leading to embryonic membrane remodeling prior to birth and to test inhibitors of MMPs for their ability to prevent collagenolysis and fetal membrane rupture.

We report here that rat amnion type IV collagens are composed primarily of alpha1(IV) and alpha2(IV) chains. Amnion basement membrane collagens were more sensitive to degradation by collagenases than were adult rat kidney basement membrane collagens, which are enriched in alpha3(IV), alpha4(IV), and alpha6(IV) chains. Amnion type IV collagen content per unit of protein was markedly reduced by Day 21 of pregnancy, the day of delivery. Increased amnion levels of matrix metalloproteinase (MMP)-2 and MMP-9, gelatinases that degrade type IV collagen, were found by Day 21, suggesting that collagen breakdown was responsible, in part, for the decline in amnion type IV collagen. Infection of organ cultures of Day 18 rat amnions with a recombinant adenovirus expressing MMP-9 (AdMMP-9) caused release of collagen fragments detected as hydroxyproline in the culture fluid, amnion cell detachment, and apoptosis. The AdMMP-9-induced apoptosis was prevented by the MMP inhibitor batimastat. These findings suggest that MMPs are implicated in anoikis and apoptotic death of amnion cells, and may be part of a complex program of fetal membrane remodeling that occurs before delivery.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.

Premature rupture of fetal membranes can harm infant and mother. It is unclear whether structural changes predispose these membranes to breaking. We thus assessed rat visceral yolk sac placenta (VYSP) and amnion by light and by transmission electron microscopy on Days 18-21 of gestation. Light microscope sections were stained for connective tissue (extracellular matrix) components: collagen, glycoprotein, and glycosaminoglycans/proteoglycans. Some tissue was incubated with chondroitinase ABC. We observed that fetal membranes became increasingly fragile, rupturing readily on Day 21. On Days 18-20, the two epithelial layers of the capsular VYSP were separated by a well-developed, well-vascularized connective tissue layer that stained intensely for all matrix components studied; on Day 21, the connective tissue layer was thinner, moderately stained, and less vascularized. On Days 18-20, the two cellular layers of the amnion were separated by a narrow, compact connective tissue layer that stained modestly for all matrix components; on Day 21, this area was widened and stained faintly. Transmission electron microscopy showed that collagen fibrils of the amnion were abundant, closely packed, and well organized on Days 18-20, whereas on Day 21 they were few in number, widely spaced, and disorganized. Similar changes were present after incubation with chondroitinase ABC. In addition, amniotic epithelial cells were moribund and delaminating, basal laminae were deteriorating or absent, and few cells were at the outer surface of the amnion. All changes preceded parturition. We conclude that the structural integrity of rat fetal membranes is impaired before birth through the loss of connective tissue components and cells, changes that presumably underlie membrane rupture. Lastly, the similarity of structural changes in rat and human fetal membranes point to the potential usefulness of the rat model.

The fetal membranes undergo striking changes in structure before delivery that involve catabolism of the extracellular matrix. To investigate the role of specific enzymes in this process, we examined gelatinase activities in rat amnion, visceral yolk sac placenta, and placenta and amniotic fluid between Days 18-21 of pregnancy. Matrix metalloproteinase (MMP)-2 was present in amnion on all days, and its activity increased slightly on Day 21. The 92-kDa gelatinase, MMP-9, was not detected on Days 18-20 but appeared by the morning of Day 21. There was a marked increase in MMP-9 mRNA in the amnion on Day 20, preceding the appearance of MMP-9 activity. Western blotting confirmed an increase in MMP-9 protein in amnion on Day 21. MMP-2 and MMP-9 activities were detected in extracts of whole yolk sac placenta, placenta, and amniotic fluid, but there were no striking changes in these gelatinases between Days 18 and 21. However, the capsular regions of the visceral yolk sac placentae, which thin and rupture during labor, did show higher MMP-9 activity on Day 21 than on Days 18 and 20. We suggest that the striking increase in MMP-9 expression in amnion and possibly the capsular region of the visceral yolk sac placenta approximately 12 h prior to delivery is responsible, in part, for the alterations in the structure of these fetal membranes before parturition.

To determine whether specific collagenolytic enzymes are expressed in human fetal membranes with labor, we examined gelatinase activity in extracts of amniochorion by zymography. The 92-kd gelatinase (MMP-9) was barely detectable in extracts of fetal membranes before the onset of labor but was readily demonstrable in extracts prepared from membranes isolated from laboring women or membranes collected immediately after delivery. In contrast, the 72-kd gelatinase (MMP-2) was detectable in extracts from pre- and post-labor membranes. Ethylenediaminetetracetic acid and the tissue inhibitor of metalloproteinases, TIMP-1, inhibited the gelatinase activities detected by zymography, confirming that the enzymes are metalloproteinase. Assay of amniochorion gelatinase activity using a radiolabeled denatured collagen substrate revealed a more than twofold increase in activity comparing pre-labor with post-labor fetal membrane extracts. A function-blocking anti-MMP-9 monoclonal antibody inhibited pre-labor membrane gelatinase activity by approximately 11.5%, which was only slightly greater inhibition than observed with irrelevant monoclonal antibodies. However, post-labor membrane gelatinase activity was reduced by 53% by the function-blocking antibody, indicating that MMP-9 is a major contributor to the increased gelatinase activity extractable from post-labor membranes. Western blot analyses demonstrated increased MMP-9 protein in amniochorion extracts after onset of labor. MMP-9 protein and mRNA were co-localized in amnion epithelium, underlying macrophages and chorion laeve trophoblast and decidual cells after labor. We conclude that 1) MMP-9 activity and protein in human amniochorion increases with labor and 2) MMP-9 is expressed by amnion epithelium, macrophages and chorion laeve trophoblast and decidual cells. The increased expression of MMP-9 may result in degradation of the extracellular matrix of the fetal membranes and facilitate their rupture under both physiological and pathological conditions.