Down regulation of the expression of the p110, p85 and p55 subunits of phosphatidylinositol 3-kinase during colon cancer cell anchorage-independent growth.

We have found that analogues of phosphatidylinositol modified at the 3'-myo-inositol position prevent phosphorylation by phosphatidylinositol 3-kinase (PI3-kinase) inhibit the anchorage-independent growth of HT-29 human colon adenocarcinoma cells as colonies in soft agarose but have no effect on monolayer growth on a plastic surface. Examination of an incomplete differential display library revealed 3 genes whose expression was increased and 11 genes whose expression was decreased or absent in HT-29 cells growing as colonies compared to monolayer growth. One of the cDNAs corresponding to an mRNA that was expressed only in cells growing as a monolayer was a 126 bp fragment that had 98% identity with a fragment of mRNA for human p55PIK PI3-kinase regulatory subunit. The down regulation of p55PIK gene expression in HT-29 cells growing as colonies was confirmed by RT-PCR using p55PIK-specific oligonucleotide primers. RT-PCR and Northern hybridisation were used to show that the expression of the p110 catalytic subunit and the p85 regulatory subunit of PI3-kinase were also down regulated in HT-29 cells growing as colonies. We measured a decrease of approximately 25% in total PI3-kinase activity of the HT-29 cells grown in soft agarose compared to HT-29 cells grown as monolayer. The results suggest that p110 PI3-kinase activity may be limiting in cells showing anchorage independent-growth which could explain their increased sensitivity, compared to cells in monolayer culture, to inhibitors of PI3-kinase signalling.

The expression of the molecular chaperone calnexin is decreased in cancer cells grown as colonies compared to monolayer.

Differential display was used to identify genes which were differentially expressed when HT-29 human colon adenocarcinoma cells were grown as monolayers attached to plastic or as colonies in soft agarose. One of the gel bands differentially displayed corresponded to a 171 bp fragment that showed 99% identity with a sequence of mRNA for human calnexin. The decrease in calnexin gene expression by HT-29 cells growing as colonies in soft agarose was confirmed by reverse transcriptase-PCR (RT-PCR) using calnexin-specific primers. We also used RT-PCR to show that the expression of calnexin was decreased in HT-29 cells and MCF-7 human breast adenocarcinoma cells growing in suspension in poly(hydroxyethyl methacrylate)-coated wells compared to cells growing as monolayers. The results suggest that there is a signal for the up-regulation of calnexin expression when cells contact a substrate which allows cell adhesion.

Initiation of heterotopic osteogenesis in primates after chromatographic adsorption of osteogenin, a bone morphogenetic protein, onto porous hydroxyapatite.

The chromatographic adsorption of partially purified osteogenin, a bone morphogenetic protein (BMP), onto porous hydroxyapatites for the construction of an osteogenic delivery system was investigated. Osteogenin fractions, with osteogenic activity in rats, were chromatographically adsorbed onto porous hydroxyapatites and implanted intramuscularly in baboons (Papio ursinus). Histology showed extensive bone differentiation within the porous spaces as early as 30 days after implantation in baboons. The unbound eluate from the porous hydroxyapatites showed lack of osteogenic activity when bioassayed in rats. These findings in primates may help to construct custom made porous ceramic as carriers for BMPs resulting in predictable bone formation for treatment of human bone defects.

Reconstruction of the bone--bone marrow organ by osteogenin, a bone morphogenetic protein, and demineralized bone matrix in calvarial defects of adult primates.

Information concerning the efficacy of osteogenin, a bone morphogenetic protein, and demineralized bone matrix in orthotopic sites in nonhuman primates is a prerequisite for potential clinical application in humans. After exposure of the calvaria, 84 cranial defects, 25 mm in diameter, were prepared in 26 adult male baboons (Papio ursinus). Defects were implanted with insoluble collagenous bone matrix (ICBM, the inactive collagenous residue after dissociative extraction of bone matrix with 4 M guanidine hydrochloride) reconstituted with osteogenin fractions isolated from baboon bone matrix by chromatography on heparin-Sepharose and hydroxyapatite-Ultrogel (Og Hep-HA) or osteogenin further purified using Sephacryl S-200 gel filtration chromatography (Og S-200). Baboon osteogenin with the highest biologic activity in a rodent bioassay, as determined by alkaline phosphatase activity, calcium content, and histologic analysis, was used for orthotopic implantation in baboons. Additional defects were implanted with baboon demineralized bone matrix (DBM) or ICBM without osteogenin as control. Defects also were grafted with corticocancellous bone harvested from the iliac crest or left ungrafted to monitor the spontaneous regeneration potential of the adult baboon calvaria. Undecalcified bone sections at 7 microns were prepared from the harvested specimens 30 and 90 days after surgery. Histomorphometry demonstrated that Og S-200 induced copious amounts of bone and osteoid as early as day 30 (P < 0.01 versus ICBM, autogenous grafts and untreated defects). At day 90, in implants of Og S-200, Og Hep-HA, and DBM, bone and marrow formation was extensive, culminating in complete regeneration of the craniotomies. In implants of DBM, bone formed with an intervening phase of cartilage development. This provides the phenotypic evidence of endochondral bone differentiation by induction in defects of membranous calvarial bone in adult primates. These results establish the potential therapeutic application of osteogenin and demineralized bone matrix for the architectural reconstruction of the bone-bone marrow organ in humans.

Initiation of bone regeneration in adult baboons by osteogenin, a bone morphogenetic protein.

Osteogenin, and related bone morphogenetic proteins, induce endochondral bone differentiation through a cascade of events which include formation of cartilage, hypertrophy and calcification of the cartilage, vascular invasion, differentiation of osteoblasts, and formation of bone. These events have been studied in a postnatal model of bone development in rodents. Information concerning the morphogenetic potential of osteogenin in primates is a prerequisite for potential clinical application in man. The efficacy of allogeneic osteogenin in primates was investigated in both extraskeletal and skeletal sites in 19-Chacma baboons (Papio ursinus). Osteogenin was isolated from demineralized baboon bone matrix and purified by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Protein fractions with a molecular mass range of 26-42 kDa induced cartilage and bone differentiation in the subcutaneous space of rats. Final purification to homogeneity was obtained by electroendosmotic elution from a preparative sodium dodecyl sulphate (SDS) polyacrylamide gel, resulting in a single band on a SDS-polyacrylamide gel with an apparent molecular mass of 30-34 kDa, with biological activity in rats. The osteoinductive potential of osteogenin in primates was tested first in intramuscular sites in baboons and found to be active. The bone regeneration potential was investigated in nonhealing calvarial defects surgically prepared in adult male baboons. Baboon osteogenin induced complete regeneration of the cranial wound. These findings in adult primates establish a primary role for osteogenin in initiation and promotion of osteogenesis, and imply a potential therapeutic application based on cell biology of extracellular matrix-cell interactions.