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Biomedical subjects

M E Stearns

Publications and source records attributed to M E Stearns.

At least 37 records · Page 2Linked to original sources
PubMed1996

Immunohistochemical studies of activated matrix metalloproteinase-2 (MMP-2a)expression in human prostate cancer.

Previous immunolabeling studies have indicated that increased expression of the matrix metalloproteinase-2 (MMP-2) zymogen is associated with an increased Gleason score for human prostate cancer. In the accompanying paper, we have found by immunoblotting and ELISA that the MMP-2 enzyme (termed MMP-2a) is expressed in prostate cancer and that increased expression is associated with progression. Monoclonal antibodies specific for MMP-2a were used to investigate the expression of MMP-2a in human prostate tissue sections of benign and malignant cancers. Immunohistochemistry indicated that MMP-2a expression was undetectable in fetal (n = 4), benign (n = 11), and low Gleason score 4 (n = 8) tissue. MMP-2a was faintly expressed (+) in cancer assigned Gleason scores 5 (n = 20) and 6 (n = 13). In comparison, MMP-2a was expressed at an intermediate level (++) in tissues of Gleason score 7 (n = 24), and at a intense level ( to +) in tissues of score 8 (n = 48), 9 (n = 9) and 10 (n = 35) and in lymph node metastases (n = 10). These observations were confirmed by quantitative Computer Assisted Imaging Analysis. In general, MMP-2a was primarily expressed by the glandular epithelial cells, and in high Gleason score 10 specimens (n = 25/35) there was clear evidence of MMP-2a localization at the cell surface. These data suggest that increased MMP-2a expression may be associated with malignant progression and metastases.

Enzyme Activation↗
PubMed1996

Evidence for increased activated metalloproteinase 2 (MMP-2a) expression associated with human prostate cancer progression.

Immunolabeling studies have previously indicated that increased expression of the 72-kDa matrix metalloproteinase 2 (MMP-2) is associated with human prostate cancer progression. It is not known if the enzymatically active MMP-2 is expressed in prostate cancer and if increased expression is associated with progression. Monoclonal antibodies specific for the activated MMP-2 molecule (MMP-2a, 66 kDa) were used (along with previously developed MMP-2 antibodies) to investigate the expression of MMP-2a and MMP-2 in human prostate tissue extracts. SDS-PAGE, Western blots, and zymography indicated that MMP-2a expression was undetectable in normal prostate (n = 6), benign prostatic hyperplasia (n = 9), and in prostate cancer of low Gleason score (GS) 4 (n = 11). MMP-2a was expressed in prostate cancer of increased GS (n = 37) and in lymph node metastases (n = 7). Quantitative ELISAs of human prostate cancer tissue extracts revealed that the levels of MMP-2 and MMP-2a per microgram of protein increased in prostate cancer tissues of increased GS (n = 48). MMP-2a levels were also high in prostatic lymph node metastases, but MMP-2 was not expressed or was barely detectable in these tissues. The molar ratios of MMP-2a to MMP-2 increased from 0 to 6.23 in tissues of GS 4 to 10, respectively. We conclude that significant increases in MMP-2a are associated with the malignant progression of prostate cancer and with tumor cell metastases to lymph nodes.

Blotting, Western↗
PubMed1996

Cytokine regulation of the matrix metalloproteinases and their inhibitors in human papillomavirus-18 transformed human prostatic tumor cell lines.

Cytokines may play a critical role in influencing the invasive and metastatic behavior of advanced cancers. To investigate the influence of cytokines on tissue inhibitor of metalloproteinase (TIMP) and matrix metalloproteinase (MMP) expression we have established cultures from prostate tissues of low Gleason sum 5 and high Gleason sum 10 cancers. We have examined the influence of different cytokines (interleukin [IL]-10, IL-4, IL-6, IL-2, and interferon-gamma) on TIMP-2, MMP-2, and MMP-9 protein and mRNA expression in human papillomavirus (HPV)-18 immortalized human prostate cell lines derived from the primary cultures. Western blot and northern blot analysis revealed that IL-10, IL-6 and IL-4 all upregulated TIMP-1 expression after 16-36 h. In contrast, IL-10 and IL-4 (but not IL-6) downregulated MMP-2 mRNA and protein levels to different degrees over 24-36 h. The levels of TIMP-2 and MMP-9 protein and mRNA were not influenced substantially by any of the cytokines. Also, IL-2 and interferon-gamma had little or no effect on any of these genes. In sum, the data showed that IL-10 (or IL-4) upregulated TIMP-1 and coordinately downregulated MMP-2 expression. Thus, cytokines might control the molar ratio of TIMP-1 and MMP-2 to influence the level of protease activity and perhaps the invasive behavior of malignant cells in vivo.

Adenocarcinoma↗
PubMed1996

Effects of alendronate and taxol on PC-3 ML cell bone metastases in SCID mice.

The combined influence of alendronate, a bisphosphonate compound, and taxol on the establishment and growth of human PC-3 ML subclones injected intravenously via the tail vein in SCID mice was investigated. The pretreatment of SCID mice with alendronate (0.04-0.1 mg/kg twice weekly or 0.1 mg/kg weekly) partially blocked the establishment of bone metastases by human PC-3 ML cells and resulted in tumor formation in the peritoneum and other soft tissues. However, alendronate pretreatment of mice (0.1 mg/kg twice weekly or weekly) and dosing along with taxol (10-50 mg/kg/day, twice weekly, or weekly) blocked the growth of PC-3 ML tumors in the bone marrow and soft tissues in a statistically significant manner and improved survival rates significantly (p < 0.001) by 4-5 weeks. ELISAs and zymography of matrix metalloproteinase production in vitro and in vivo showed that alendronate and taxol alone partially inhibited metalloproteinase production, but that taxol in combination with alendronate totally blocked protease production and release. The combined activities of alendronate and taxol appeared to inhibit the establishment and growth of tumors in SCID mice, perhaps, in part, as a result of inhibition of protease production and release.

Alendronate↗
PubMed1995

Keratinocyte growth factor and receptor mRNA expression in benign and malignant human prostate.

We have examined whether keratinocyte growth factor (KGF) and its receptor are expressed in normal, fetal, and prostate cancer cells since KGF may play a role in the growth of adenocarcinomas. In situ hybridization studies with digoxigenin-labeled oligonucleotides (anti-sense and sense controls) were employed to examine KGF and KGF receptor mRNA expression in prostate cancer. We found that the KGF and KGF receptor genes were faintly expressed in the stromal and epithelial cells, respectively, in both fetal (n = 6) and normal adult prostate (n = 6) tissues examined. In 10 benign prostatic hyperplasias (BPH), and in low- and high-grade prostatic carcinoma (32 total), both the KGF gene and the receptor mRNA were expressed in the glandular epithelial cells. KGF was also expressed by the stromal cells in BPH and low-grade carcinoma. Computer assisted system analysis indicated that the intensity of epithelial labeling by both probes was increased in high Gleason score carcinomas ( > 8) and in metastatic nodules. We interpret the data to mean that the paracrine loop in normal prostate may be replaced by an autocrine loop in BPH and adenocarcinomas.

Adult↗
PubMed1995

IL-10 blocks collagen IV invasion by "invasion stimulating factor" activated PC-3 ML cells: upregulation of TIMP-1 expression.

We have previously shown that a 78-kDa "invasion stimulating factor" (ISF) triggers collagenase IV (MMP-2) secretion and the invasive behavior of metastatic PC-3 ML subclones in modified Boyden chamber assays [Stearns, M. E.; Stearns, M. Autocrine factors, type IV collagenase secretion and prostatic cancer cell invasion. Cancer Metastasis Rev. 12:39-52; 1993. Wang, M.; Stearns, M.; Stearns, M. E. Identification of the receptor for a novel M(r) 78,000 "invasion stimulating factor" from metastatic human prostatic PC-3 ML clones. Cancer Res. 54:2492-2495; 1994.]. Recently, we have shown that interleukin 10 (IL-10) preferentially stimulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in these cells [Wang, M.; Stearns, M. E. Characterization of a novel TIMP-1 enhancer element. J. Biol. Chem., submitted.]. In this paper, we report that IL-10 (20-40 ng) can inhibit the invasion stimulatory effects of ISF (30-60 ng) on PC-3 ML cells. "Checkerboard analysis" with modified Boyden chambers (precoated with 10 and 100 micrograms collagen IV) shows that IL-10 inhibits the stimulatory effects of ISF on both cell motility and chemoinvasion processes. In support of these data, exogenously supplied TIMP-1 (10 micrograms/ml) and collagenase antibodies (1:200 dilution) both completely blocked invasion. Quantitative ELISAs comparing the molar ratios of TIMP-1:MMP-2 and TIMP-2:MMP-2 further demonstrate that IL-10 (10-40 ng) preferentially activates TIMP-1 secretion to increase the molar ratio of TIMP-1:MMP-2 in the presence of increasing amounts of ISF (0-60 ng). IL-10 did not elevate TIMP-2 secretion or influence the molar ratio of TIMP-2:MMP-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cytokine (IL-10, IL-6) induction of tissue inhibitor of metalloproteinase 1 in primary human prostate tumor cell lines.

Northern blots and scintillation counting showed that tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA was expressed by low passage, primary epithelial cultures (n = 5) of low-grade human prostatic carcinoma. TIMP-1 mRNA levels were normally low in the primary cell lines, but were inducible by interleukin (IL) 10 and 6. Dose and time-course studies indicated that IL-10 was the most potent stimulator of TIMP-1 expression. Cycloheximide blocked the effects of IL-10 in a reversible manner. In situ hybridization assays with TIMP-1 oligonucleotide antisense probes confirmed the northern blot results and indicated that IL-10 preferentially stimulated TIMP-1 mRNA synthesis. We suggest that IL-10, and to a lesser extent IL-6, may normally influence TIMP-1 expression by human prostatic epithelial cells.

Blotting, Northern↗
PubMed1995

Taxol reduces circulating tumor cells to prevent bone metastases in SCID mice.

The correlative effects of taxol on the reduction of circulating PC-3 ML human prostatic tumor cells and bone metastasis have been examined in SCID mice. Normally, following injection of 2 x 10(5) cells i.v., the circulating levels in peripheral blood drop by about 50 and 100%, after 8 and 24 h, respectively. In contrast, in taxol-treated mice (40-60 mg/m2/injection given 0, 3, 7 and 23 h following injection of the cells) the numbers of circulating human prostatic PC-3 ML tumor cells were reduced by 100% at 8 h. In similar experiments were mice were injected with taxol 2 h prior to injecting the cells, dosages of 40 and 60 mg/m2/injection reduced circulating tumor cells about 91 and 100%, respectively, by 8 h. Alternatively, if PC-3 Ml cells were pretreated with taxol (0.5 and 1.0 microM for 8 and 24 h) prior to injection, tumor cell clearance by 7 h was also significantly increased (80-100%). Correlative studies showed that the incidence of bone metastases (observed after 40 days) was reduced significantly (a) in mice treated with 40 and 60 mg/m2/injection (i.e. from 73-80% in controls to 15-0% in treated mice) and (b) in mice injected with PC-3 ML cells pre-exposed to 0.5-1.0 microM taxol for 7 h. Immunofluorescence studies with tubulin antibodies showed that the microtubules were disrupted in cells exposed to taxol in vivo and in vitro under conditions that significantly increased cellular clearance from the blood. Taken together, the data suggests that taxol at nontoxic dosages (to mice) can prevent metastases by directly reducing the circulating levels of tumor cells.

Animals↗
PubMed1994

Identification of the receptor for a novel M(r) 78,000 "invasion stimulating factor" from metastatic human prostatic PC-3 ML clones.

A M(r) 78,000 protein (reduced), termed invasion stimulating factor (ISF), was purified from the conditioned medium of a bone metastasizing human prostatic PC-3 ML clone (M. E. Stearns and M. Stearns. Cancer Metastasis Rev., 12: 39-52, 1993). Scatchard analysis and affinity cross-linking studies revealed that the producer PC-3 ML cells expressed a receptor binding site (M(r) approximately 115,000). We found a Kd approximately 425 pM and about 22,000 sites/cell. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms confirmed that the PC-3 ML cells expressed the receptor, whereas the ISF non-producing, noninvasive PC-3 clones (i.e., 3-4 x N.I. PC-3 cells) failed to express the ISF receptor. We conclude that a unique ISF autocrine loop characterizes the bone metastatic PC-3 ML cells.

Bone Neoplasms↗
PubMed1994

Immunohistochemistry analysis of platelet-derived growth factor A and B chains and platelet-derived growth factor alpha and beta receptor expression in benign prostatic hyperplasias and Gleason-graded human prostate adenocarcinomas.

The purpose of this study was to determine whether changes in the expression of platelet-derived growth factor (PDGF) and its receptors were associated with prostate cancer. Peroxidase-anti-peroxidase-immunoperoxidase labeling was used to detect the A and B chains and alpha and beta receptors of PDGF in 5 benign prostatic hyperplasias and 13 human prostate adenocarcinomas (Gleason grades 2 to 9). In all 5 benign prostatic hyperplasias, none of the PDGF antibodies (even at high titers, 1:50 dilutions) labeled the epithelial or the stromal cells. In adenocarcinomas, the A chain and alpha receptor antibodies labeled both epithelial and stromal cells. The intensity of the staining was relatively high in low Gleason grade (<6) and low in high Gleason grade (>7) tissue; however, the B chain and beta receptor antibodies failed to label either epithelial or stromal cells in any of the 13 adenocarcinomas. The data suggest that PDGF A and alpha receptor genes may be preferentially turned on in epithelial and stromal prostate tumor cells.

Adenocarcinoma↗
PubMed1994

Decreased expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in invasive cell clones derived from human prostate and breast tumor cells.

The importance of receptors involved in the clearance of proteases and protease/inhibitor complexes in tumor invasion is unknown. We studied the expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR), the major receptor involved in the clearance of protease/inhibitor complexes, in invasive and noninvasive subclones derived from tumor cells. The receptor activity was 2- to 3-fold lower in invasive subclones compared to noninvasive subclones derived from human prostate PC-3 and DU 145 and melanoma A2058 cells. The receptor activity was decreased in breast cancer (MCF-10A) cells transfected with mutated Ha-ras compared to nontransfected cells. Furthermore, invasive cells derived from ras-transfected MCF-10A cells expressed lower levels of receptor compared to their non-invasive counterparts. These studies indicate a correlation between invasive phenotype and low receptor expression in different tumor cells. Experiments were performed to understand two possible mechanisms (decreased transcription of the receptor and increased transcription of an inhibitory protein) for the decreased cell-surface expression of the receptor in invasive cells. The decreased expression in invasive subclones was due to 2- to 3-fold lower levels of LRP/alpha 2-MR mRNA in all cells. In invasive PC-3 subclones, but not in DU 145, A2058, and MCF-10A subclones, 2-fold higher levels of the 39 kDa receptor-associated protein (an inhibitor of the receptor) mRNA were observed. These studies showed that the decreased expression of LRP/alpha 2-MR activity in invasive subclones was generally correlated with the decreased steady-state mRNA levels of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Immunoassays of the metalloproteinase (MMP-2) and tissue inhibitor of metalloproteinase (TIMP 1 and 2) levels in noninvasive and metastatic PC-3 clones: effects of taxol.

We have selected and subcloned bone metastatic (PC-3 ML) and noninvasive, nonmetastatic (3 x N.I.) lines from human prostatic PC-3 parent cells. In this paper, we have compared relative levels of MMP-2 and TIMP-1 and 2 in PC-3 tumors grown in severe combined immunodeficient or SCID mice. Dot blots with polyclonal antibodies specific for MMP-2, TIMP-1 and TIMP-2 revealed that the MMP-2 levels were high in subcutaneously grown PC-3 ML clones but low in 3 x N.I. clones at days 20 and 40. The TIMP-1 levels were inversely proportional to MMP-2 in the two types of clones, respectively. The TIMP-2 levels were similar in both clones at days 20 and 40. Gelatin zymograms confirmed that PC-3 ML tumors contained MMP-2 (and not MMP-9) subcutaneously or in bone metastases in SCID mice. Slot blots of PC-3 ML bone tumors comparing MMP-2 and TIMP-1 levels showed at days 10, 15, 17, 19, 21, and 25 that the ratio of MMP-2 to TIMP-1 increased, especially at about day 21, when extensive secondary metastases to the peritoneal cavity occurred. The levels of TIMP-2 remained constant. Quantitative ELISAs confirmed the blotting data and showed that taxol blocked MMP-2 but not TIMP-1 production in these advanced tumors. We conclude that highly metastatic PC-3 ML variants contained relatively high levels of MMP-2 and low amounts of TIMP-1.

Animals↗
PubMed1993

Liarozole and 13-cis-retinoic acid anti-prostatic tumor activity.

Liarozole fumarate (R85,246), a novel benzimidazole derivative, reduced s.c. and bone metastasis tumor growth by the androgen-independent PC-3ML-B2 human prostatic carcinoma clone in SCID mice. The drug inhibited cell invasion of Matrigel in Boyden chamber chemotactic assays and the secretion of type IV collagenase. In vitro, liarozole failed to inhibit cell proliferation and cell attachment to various substrates (Matrigel, laminin, type IV collagen, and fibronectin). In vivo, the drug also blocked type IV collagenase production in established s.c. tumors. Liarozole has been postulated by others (R. De Coster, W. Wouters, R. Van Ginckel, D. End, et al. J. Steroid Biochem. Mol. Biol., 43: 197-201, 1992) to inhibit retinoic acid catabolism. Our data indicate that liarozole treatment can increase the tumor retinoic acid levels in vivo. Studies of retinoic acid revealed that the drug independently reduced tumor growth in vivo and inhibited cell invasion of Matrigel and the secretion of collagenase IV. Surprisingly, liarozole and retinoic acid failed to exhibit measurable synergistic activity both in vitro and in vivo. Taken together these data suggest that liarozole might inhibit retinoic acid catabolism in vivo and consequently have significant therapeutic value as an anti-prostatic tumor agent.

Animals↗
PubMed1993

Type IV collagenase (M(r) 72,000) expression in human prostate: benign and malignant tissue.

The expression of type IV collagenase (M(r) 72,000) has been examined in tissues from patients with benign prostatic hyperplasia (6 patients) and varying Gleason grades of malignant prostate cancer (18 patients). Immunoperoxidase labeling indicated that expression of the type IV collagenase was weak or nonexistent in benign tissue but consistently strong in the glandular and ductal epithelial cells of prostate tumors diagnosed at Gleason grades 1-8. In moderate to advanced cancer (i.e., Gleason grades 2 to 8), invasive tumor foci in the stromal tissue produced relatively modest amounts of type IV collagenase. The normal stromal tissue (i.e., fibroblasts) uniformly failed to produce detectable levels of type IV collagenase in the 24 patients examined. Northern and quantitative slot blot hybridization assays demonstrated that collagenase type IV mRNA levels were low in benign tissue and high in malignant tumors. In contrast, the stromal cells did not express significant amounts of type IV collagenase mRNA. Enzyme-linked immunosorbent assays demonstrated that the amounts of type IV collagenase protein correlated directly with the mRNA levels in the tumor tissue. The studies suggest that type IV collagenase may be selectively overexpressed by malignant, preinvasive prostatic epithelial cells.

Blotting, Southern↗
PubMed1993

Autocrine factors, type IV collagenase secretion and prostatic cancer cell invasion.

Motility factors play a major role in tumor cell invasion and metastases. The biochemical properties of various motility factors; the receptor mediated mechanism of action; the role of microtubules; the potential influence of oncogenes; and the influence of motility factors on type IV collagenase secretion and invasion are discussed. We report on expression of a 70 kDa motility factor, termed invasion stimulating factor (ISF), in human prostatic PC-3 sublines. Boyden chamber chemotactic assays and measurements of type IV collagenase synthesis and secretion suggest that an ISF-receptor dependent mechanism influences tumor cell invasion and protease secretion. Taken together, the evidence that autocrine motility factors play an essential role in tumor cell invasion and metastases is compelling.

Animals↗
PubMed1992

Taxol blocks processes essential for prostate tumor cell (PC-3 ML) invasion and metastases.

We have examined the antimetastatic effects of taxol on a PC-3 human prostatic tumor variant (PC-3 ML) which metastasizes to the lumbar vertebrae in severe combined immunodeficiency-carrying (SCID) mice. Immunofluorescence labeling indicated that taxol (0.5 to 1.0 microM for 6 h) produced an abnormal bundling of microtubules in a dosage-dependent manner. Slot blotting and gelatinase assays revealed that taxol inhibited secretion of the M(r) 72,000 and M(r) 92,000 type IV collagenases plus a M(r) 57,000 gelatinase. Radioimmunoprecipitation measurements confirmed that the drug inhibited both the secretion and the synthesis of the M(r) 72,000 collagenase. Taxol also blocked total protein secretion but did not influence total protein synthesis or turnover. Boyden chamber chemotactic studies further showed that taxol (0.5 to 1.0 microM) inhibited invasion of Matrigel. More importantly, studies in SCID mice demonstrated that taxol (50 to 250 mg/m2/day) blocked the establishment, growth, and long-term survival of PC-3 ML cells.

Alkaloids↗
PubMed1991

Regulation of kinesin expression and type IV collagenase secretion in invasive human prostate PC-3 tumor sublines.

PC-3 human prostatic tumor sublines have been previously isolated which exhibit striking differences in their invasive and metastatic phenotypes. This work has been extended here to measure and compare the levels of kinesin, a microtubule-dependent translocator molecule, in the PC-3 sublines. Western blots, slot blots, radiolabeling, and immunoprecipitation analysis showed that kinesin was expressed in the highly invasive and metastatic sublines at levels which were elevated above the base-line levels observed in the parent PC-3 cells. In comparison, kinesin was not expressed in detectable amounts in the noninvasive cell lines. The conditioned medium of the metastatic PC-3 sublines contained a heat- and trypsin-sensitive protein which exhibited a dosage-dependent capacity to stimulate increased kinesin expression, type IV collagenase secretion, and invasion of Matrigel by the metastatic sublines. The noninvasive sublines failed to secrete a similar stimulatory factor(s) or respond to the conditioned medium of metastatic sublines. Various growth factors and cytokines tested (platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, formylmethionineleucinephenylalanine) had no significant effect on either kinesin expression or protease secretion and invasion. Pertussis toxin blocked the stimulatory effects of the conditioned medium, but other agents known to interfere with adenylate cyclase pathways (i.e., cholera toxin, forskolin, 8-bromoadenosine) failed to block stimulation. The data show for the first time that kinesin, protease secretion, and the resulting invasion process may be regulated in a coordinated manner by an autocrine factor(s) which activates G-protein-dependent processes.

3T3 Cells↗