Bruce E. Linebaugh

Phorbol Ester Activation of a Proteolytic Cascade Capable of Activating Latent Transforming Growth Factor-β A PROCESS INITIATED BY THE EXOCYTOSIS OF CATHEPSIN B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-β in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and β-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-β antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-β.

Mutant K-ras Regulates Cathepsin B Localization on the Surface of Human Colorectal Carcinoma Cells

Cathepsin B protein and activity are known to localize to the basal plasma membrane of colon carcinoma cells following the appearance of K-ras mutations. Using immunofluorescence and subcellular fractionation techniques and two human colon carcinoma cell lines - one with a mutated K-ras allele (HCT 116) and a daughter line in which the mutated allele has been disrupted (HKh-2)-we demonstrate that the localization of cathepsin B to caveolae on the surface of these carcinoma cells is regulated by mutant K-ras. In HCT 116 cells, a greater percentage of cathepsin B was distributed to the caveolae, and the secretion of cathepsin B and pericellular (membrane-associated and secreted) cathepsin B activity were greater than observed in HKh-2 cells. Previous studies established the light chain of annexin II tetramer, p11, as a binding site for cathepsin B on the surface of tumor cells. The deletion of active K-ras in HKh-2 cells reduced the steady-state levels of p11 and caveolin-1 and the distribution of p11 to caveolae. Based upon these results, we speculate that cathepsin B, a protease implicated in tumor progression, plays a functional role in initiating proteolytic cascades in caveolae as downstream components of this cascade (e.g., urokinase plasminogen activator and urokinase plasminogen activator receptor) are also present in HCT 116 caveolae.

Phorbol ester activation of a proteolytic cascade capable of activating latent TGF-β: A process initiated by the exocytosis of cathepsin B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-β in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and β-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-β antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-β.

Bone marrow-derived cathepsin K cleaves SPARC in bone metastasis

Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis.

Phospholipase A2 activity in 9,10-dimethyl-1,2-benzanthracene-induced mammary tumors of rats.

The activities of phospholipase A2 were compared in mammary glands from virgin and mid-pregnant rats and in 9,10-dimethyl-1,2-benzanthracene-induced rat mammary tumors. Enzyme activities were not different in the 150 000 x g pellet fractions of mammary gland homogenates from virgin and mid-pregnant rats, but enzyme activity in the 150 000 x g supernatant fraction was about twice as high in the homogenates from the mid-pregnant rat glands. Phospholipase A2 activities in the 150 000 x g pellet and supernatant fractions of homogenerates of growing tumor tissues were more than an order of magnitude higher than in the normal tissues. The elevated activity of phospholipase A2 in the tumor tissues may be related to their rapid rate of proliferation.

Differential tyrosyl-phosphorylation of multiple mitogen-activated protein kinase isoforms in response to prolactin in Nb2 lymphoma cells

Abstract Prolactin (PRL) stimulates mitogenesis and differentiative processes in a variety of cell types. Not all of the molecules involved in PRL signaling, which follows an initial PRL-receptor interaction, have been identified. In the present studies, PRL is shown to stimulate the differential tyrosyl phosphorylation of three isoforms (ERK-1, 2, and 4) of mitogen-activated protein kinases (MAP kinase) in a rat pre-T lymphoma cell line (Nb2). Evidence also suggests that PRL stimulates the tyrosyl phosphorylation of ERK-3, a MAP kinase isoform recently identified. When G1-arrested Nb2 cells are treated with 50 ng/ml oPRL, ERK-1 through 3 become tyrosyl phosphorylated within minutes (an indication of enzyme activation) and then become dephosphorylated within 30 min. Conversely, ERK-4 is rapidly tyrosyl phosphorylated by 5 min, and remains in this state for at least 1 hr.

Cathepsin K in the bone microenvironment: link between obesity and prostate cancer?

The skeleton is the most common site of metastasis in patients with advanced prostate cancer. Despite many advances in targeting skeletal metastases, the mechanisms behind the attraction of prostate cancer cells to the bone are not known. Osteoclast cathepsin K, due to its ability to effectively degrade bone matrix collagen I, has been implicated in colonization and growth of prostate tumours in the bone. Identification of new cathepsin K substrates in the bone microenvironment and the recent findings demonstrating its involvement in obesity and inflammation suggest additional roles for this enzyme in skeletal metastases of prostate cancer.

Characteristics of the insulin stimulation of DNA, RNA and protein metabolism in cultured human mammary carcinoma cells

In a mammary gland cell line (MCF-7) of human origin, insulin stimulated the rates of RNA, DNA and protein biosynthesis. These effects were observed with concentrations of insulin ranging from 10(-6) M to 10(-10) M. Enhanced rates of [3H]leucine incorporation into protein and [3H]uridine incorporation into RNA were observed within 1 h after exposing the cells to insulin. In contrast, a stimulatory effect of insulin on the rate of [3H]thymidine incorporation into DNA was only detectable following a 12--16 h incubation with insulin. Insulin also enhanced the rate of uptake of [3H]leucine, [3H]uridine, and [3H]thymidine into the MCF-7 cells. Finally, incubation with insulin increased the total amount of DNA, RNA and protein in these cells.

Hu/Mu ProtIn Oligonucleotide Microarray: Dual-Species Array for Profiling Protease and Protease Inhibitor Gene Expression in Tumors and Their Microenvironment

Proteolysis is a critical regulatory mechanism for a wide variety of physiologic and pathologic processes. To assist in the identification of proteases, their endogenous inhibitors, and proteins that interact with proteases or proteolytic pathways in biological tissues, a dual-species oligonucleotide microarray has been developed in conjunction with Affymetrix. The Hu/Mu ProtIn microarray contains 516 and 456 probe sets that survey human and mouse genes of interest (proteases, protease inhibitors, or interactors), respectively. To investigate the performance of the array, gene expression profiles were analyzed in pure mouse and human samples (reference RNA; normal and tumor cell lines/tissues) and orthotopically implanted xenografts of human A549 lung and MDA-MB-231 breast carcinomas. Relative gene expression and “present-call” P values were determined for each probe set using dChip and MAS5 software, respectively. Despite the high level of sequence identity of mouse and human protease/inhibitor orthologues and the theoretical potential for cross-hybridization of some of the probes, >95% of the “present calls” (P < 0.01) resulted from same-species hybridizations (e.g., human transcripts to human probe sets). To further assess the performance of the microarray, differential gene expression and false discovery rate analyses were carried out on human or mouse sample groups, and data processing methods to optimize performance of the mouse and human probe sets were identified. The Hu/Mu ProtIn microarray is a valuable discovery tool for the identification of components of human and murine proteolytic pathways in health and disease and has particular utility in the determination of cellular origins of proteases and protease inhibitors in xenograft models of human cancer. (Mol Cancer Res 2007;5(5):443–54)

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.