F. Marini

BACKGROUND: High concentrations of interferon beta (IFN-beta) inhibit malignant cell growth in vitro. However, the therapeutic utility of IFN-beta in vivo is limited by its excessive toxicity when administered systemically at high doses. Mesenchymal stem cells (MSC) can be used to target delivery of agents to tumor cells. We tested whether MSC can deliver IFN-beta to tumors, reducing toxicity. METHODS: Human MSC were transduced with an adenoviral expression vector carrying the human IFN-beta gene (MSC-IFN-beta cells). Flow cytometry was used to measure tumor cell proliferation among in vitro co-cultures of MSC-IFN-beta cells and human MDA 231 breast carcinoma cells or A375SM melanoma cells. We used a severe combined immunodeficiency mouse xenograft model (4-10 mice per group) to examine the effects of injected MSC-IFN-beta cells and human recombinant IFN-beta on the growth of MDA 231- and A375SM-derived pulmonary metastases in vivo and on survival. All statistical tests were two-sided. RESULTS: Co-culture of MSC-IFN-beta cells with A375SM cells or MDA 231 cells inhibited tumor cell growth as compared with growth of the tumor cells cultured alone (differences in mean percentage of control cell growth: -94.0% [95% confidence interval [CI] = -81.2% to -106.8%; P<.001] and -104.8% [95% CI = -82.1% to -127.5%; P<.001], respectively). Intravenous injection of MSC-IFN-beta cells into mice with established MDA 231 or A375SM pulmonary metastases led to incorporation of MSC in the tumor architecture and, compared with untreated control mice, to prolonged mouse survival (median survival for MDA 231-injected mice: 60 and 37 days for MSC-injected and control mice, respectively [difference = 23.0 days (95% CI = 14.5 to 34.0 days; P<.001]; median survival for A375SM-injected mice: 73.5 and 30.0 days for MSC-injected and control mice, respectively [difference = 43.5 days (95% CI = 37.0 to 57.5 days; P<.001]). By contrast, intravenous injection of recombinant IFN-beta did not prolong survival in the same models (median survival for MDA 231-injected mice: 41.0 and 37.0 days for IFN-beta-injected and control mice, respectively [difference = 4 days, 95% CI = -5 to 10 days; P = .308]; median survival for A375SM-injected mice: 32.0 and 30.0 days for IFN-beta-injected and control mice, respectively [difference = 2 days, 95% CI = 0 to 4.5 days; P = .059]). CONCLUSIONS: Injected MSC-IFN-beta cells suppressed the growth of pulmonary metastases, presumably through the local production of IFN-beta in the tumor microenvironment. MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites.

SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.