Donald C. Paul

Mineralization and metabolic response in serially passaged adult rat bone cells.

SummaryCell populations derived from adult rat bone were grown in cell culture and characterized with respect to their morphology and response to hormones. The cells were isolated from adult rat calvaria by mechanical rather than enzymatic methods. Cultures were initiated in modified BGJb medium supplemented with fetal bovine serum. These cultures and several cloned populations derived from them retained the ability to mineralize in vitro even after extended serial passage.Cultures derived from an osteoblast-enriched population showed an initial positive cAMP response to PTH and PGE2, but not to TCT. The PTH and PGE2 responses diminished with serial passage. The PTH response was no longer measurable at passage 6, and the PGE2 response was not evident in passage 11. In one clone, the PGE2 response persisted through passage 16. Adult rat skin fibroblasts cultured similarly did not respond to PTH or TCT, but still had a significant PGE2 response through passage 21.The cultured cells formed multiple layers with localized areas of higher cell density. Mineral plaques with major diameters as great as 0.75 mm were evident in the areas of greater cell density. Less extensive mineral deposits were present throughout the culture. The mineral plaques consisted of apatite-like crystals deposited on an organic matrix. Matrix vesicles and mineralized spherules appeared to be associated with initial mineral deposition. The spherules apparently coalesced to form more complex mineralized structures. A limited amount of mineralization also was observed in rat skin fibroblast cultures.

Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1α./CXCR4- mediated migration of epitheloid carcinoma cells

Emerging evidence shows that the stromal cell–derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1α ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1α results in time-dependent Akt and extracellular signal–regulated kinase 1/2 (ERK1/2) activations. The SDF-1α–induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1α–induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1α induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1α–induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1α–induced migration of epitheloid carcinoma cells.

In vitro pharmacology of cryptophycin 52 (LY355703) in human tumor cell lines

Purpose: Cryptophycin 52 (LY355703) is a new member of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The mechanism of action of the cryptophycin class of compounds is associated with an action on microtubules. This report details the pharmacological profile of this new clinical compound in a panel of human tumor cell lines. Methods: Antiproliferative effects of cryptophycin 52 were measured indirectly by detection of the metabolic reduction of alamarBlue®. Cytoxicity was assessed by enzymatic dye activation (calcein AM) combined with dye exclusion (ethidium homodimer) and by clonogenicity assay. Cell cycle effects were evaluated using flow cytometry and fluorescence microscopy. Results: Both antiproliferative and cytotoxic effects of cryptophycin 52 were concentration- and time-dependent. IC50 values for antiproliferative activity in both solid and hematologic tumor cell lines were in the low picomolar range, and without exception, were significantly below values for the antimitotic agents paclitaxel and vinblastine. Flow cytometry and microscopic examination of tumor cells treated with cryptophycin 52 indicated that they accumulated in the mitotic phase of the cell cycle. Cryptophycin 52 was tested for its sensitivity to multidrug-resistance in several paired cell lines in which a sensitive parental line was matched with a multidrug-resistant derivative line. The resistant lines have been shown to over express Pgp and/or MRP multidrug-resistance transport factors. Compared to other antimitotic agents (paclitaxel, vinblastine, vincristine), the potency of cryptophycin 52 was shown to be minimally affected in multidrug-resistant cells compared to their sensitive parental lines. Conclusion: Cryptophycin 52 has potent antimitotic, antiproliferative and cytotoxic activity in in vitro human tumor cell models. It is significantly more potent and less sensitive to multidrug resistance mechanisms than other antimitotic antitumor agents currently used in cancer therapy. These characteristics may translate into therapeutic advantages for the clinical use of cryptophycin 52 in cancer chemotherapy.

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.

Identification of LY2510924, a Novel Cyclic Peptide CXCR4 Antagonist That Exhibits Antitumor Activities in Solid Tumor and Breast Cancer Metastatic Models

Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC50 value of 0.079 nmol/L, and inhibited SDF-1–induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1–induced cell migration with IC50 value of 0.26 nmol/L and inhibited SDF-1/CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1–stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non–Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer. Mol Cancer Ther; 14(2); 480–90. ©2014 AACR.

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

Immunocytochemical localization of plasminogen activator on porcine kidney cell strain: LLC-PK1 (LP100).

An antibody to plasminogen activator (PA) produced by the cultured cells of the pig kidney cell strain LLC-PK1 (LP100) was used to localize PA on the cells free (unattached) surface. Localization was accomplished by the unlabeled antibody enzyme method (PAP) at the light microscopic level and at the electron microsopic level. Localization was commonly more intense at cell to cell junctions and was associated with blebs and vesiculation in this area. We are proposing that membrane shedding by blebs and vesiculation may be the mechanism of PA release in the LLC-PK1 (LP100) cell strain.