Michael W. Long

Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix.

Cancer cells are able to proliferate at accelerated rates within the confines of a three-dimensional (3D) extracellular matrix (ECM) that is rich in type I collagen. The mechanisms used by tumor cells to circumvent endogenous antigrowth signals have yet to be clearly defined. We find that the matrix metalloproteinase, MT1-MMP, confers tumor cells with a distinct 3D growth advantage in vitro and in vivo. The replicative advantage conferred by MT1-MMP requires pericellular proteolysis of the ECM, as proliferation is fully suppressed when tumor cells are suspended in 3D gels of protease-resistant collagen. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices are trapped in a compact, spherical configuration and unable to undergo changes in cell shape or cytoskeletal reorganization required for 3D growth. These observations identify MT1-MMP as a tumor-derived growth factor that regulates proliferation by controlling cell geometry within the confines of the 3D ECM.

C-myc overexpression and p53 loss cooperate to promote genomic instability

p53 monitors genomic integrity at the G1 and G2/M cell cycle checkpoints. Cells lacking p53 may show gene amplification as well as the polyploidy or aneuploidy typical of many tumors. The pathways through which this develops, however, are not well defined. We demonstrate here that the combination of p53 inactivation and c-myc overexpression in diploid cells markedly accelerates the spontaneous development of tetraploidy. This is not seen with either N-myc or L-myc. Tetraploidy is accompanied by significantly higher levels of cyclin B and its associated cdc2 kinase activity. Mitotic spindle poisons accelerate the appearance of tetraploidy in cells either lacking functional p53 or overexpressing c-myc whereas the combination is additive. Restoration of p53 function in cells overexpressing c-myc causing rapid apoptosis, indicating that cells yet to become tetraploid have nonetheless suffered irreversible genomic and/or mitotic spindle damage. In the face of normal p53 function, such damage would either be repaired or trigger apoptotis. We propose that loss of p53 and overexpression of c-myc permits the emergence and survival of cells with increasingly severe damage and the eventual development of tetraploidy.

Bone marrow accessory cells regulate human bone precursor cell development.

OBJECTIVE Much remains to be learned about the intimate relationship between bone marrow and its surrounding tissue: the bone. We hypothesized that bone marrow accessory cell populations might regulate the development of human bone precursor cells. MATERIALS AND METHODS We used immunologic phenotyping, and isolation methods to fractionate subpopulations of nonadherent, low-density (NALD) human bone marrow cells. These cells were examined for their ability to support the serum-free survival, proliferation, and expression of bone proteins by highly purified populations of human bone precursor cells. Quantitative assessment of the accessory cell populations as well as human bone precursor cells phenotype was performed using multiparameter flow cytometry. Bone protein expression was evaluated by immunocytochemistry, Western analysis, and enzymatic analysis (for alkaline phosphatase activity). RESULTS Human bone marrow contains a cell population that stimulates the development of purified bone precursor cells. Feeder-layer studies demonstrate that these osteopoietic accessory cells (OACs) do not require cell-cell interaction to promote bone precursor cell development but, rather, produce soluble molecules responsible for their effects. Flow cytometric analyses reveal that bone marrow derived B cells, T cells, macrophages, natural killer cells, and endothelial cells do not produce this stimulatory factor. The (growth) factor cannot be replaced by addition of exogenous cytokines. The isolation of human transforming growth factor beta receptor type II (TGF-betaRII)-positive cells increases OAC-specific activity in bone cell ex vivo expansion cultures. Moreover, isolation of OAC bone marrow cells characterized by high TGF-betaRII expression, relatively low cellular complexity, and small size yields a population that is highly enriched for OACs. CONCLUSION We conclude that human bone marrow contains a population of OACs that are an obligate requirement for the early phases of bone cell development ex vivo.

Modulation of MDM2/p53 and cyclin-activating kinase during the megakaryocyte differentiation of human erythroleukemia cells

OBJECTIVE This study was undertaken to address the involvement of CDK activating kinase (CAK), p53, and MDM2 proteins in the mitotic arrest associated with the acquisition of a polyploid DNA content during megakaryocyte differentiation of human erythroleukemia (HEL) cells. METHODS To evaluate this mechanism we investigated HEL cells as a model system in which there is a marked increase in DNA content during megakaryocyte differentiation induced by phorbol-diesters. Specific cell-cycle phases were separated by centrifugal elutriation and SDS PAGE and Western analysis were performed to determine the relative abundance of these proteins. Kinase assays were carried out following immunoprecipitation of cellular lysates with the antibodies to the proteins. RESULTS Polyploid HEL cells show an increase in the abundance of the CAK complex proteins, CDK7 and cyclin H, and a sixfold increase in CAK-specific activity. Increased CAK activity in polyploid HEL cells follows both the downregulation of p53 protein and its decreased association with CAK complex. Consistent with the reduction of p53, polyploid HEL cells undergo a dramatic increase in MDM2 protein abundance that in turn facilitates increased interaction of this protein with p53. CONCLUSION These observations demonstrate that deregulated expression of MDM2 and p53 during megakaryocyte differentiation allow a relaxation of the control over genomic stability, allowing further replicative rounds of DNA synthesis.