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P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment.

Objective The purpose of this study was to investigate the clinical significance of DNA methylation of the human papillomavirus (HPV) genome as a prognostic biomarker for cervical intraepithelial neoplasia (CIN). Methods and Materials Clinical samples (paraffin-embedded tissues obtained by conization/hysterectomy or initial punch biopsy) were collected from patients at the Gynecologic Oncology of the Hyogo Cancer Center with informed consent. We evaluated the methylation status of the L1 gene of the HPV genome by bisulfite sequencing, calculating the methylation ratio (L1MR) as (number of methylated CpGs in the analyzed region of the L1 gene) / (number of all CpGs in the analyzed region of the L1 gene) × 100. The methylation analysis and in situ hybridization were performed with serial tissue-section slices. Results DNA methylation was observed in the L1 gene, but not in the long control region of HPV-16, -18, or the other high-risk HPV types including HPV-31, -52, and -58. L1MR was associated with the CIN grade; the median L1MR was 2.3%, 11.2%, 35.2%, and 50.0% for CIN1, CIN2, CIN3, and squamous cell carcinoma, respectively. L1MRs also seemed to indicate physical status (integrated or episomal form) of the HPV genome in the host cell. L1MR of the progression group was significantly higher than that of the regression group. Conclusions L1MR was associated with the CIN grade and indicated the HPV genome status in the host cell: high L1MR indicated HPV genome integration linked to progression from early-stage CINs, whereas low L1MR indicated an episomal HPV genome location in host cells. L1MR may be a prognostic indicator of CIN.

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow due to arterial vasodilation and augmented intrahepatic vascular resistance due to sinusoidal constriction. In this study, we examined the possible involvement of resident macrophages in the tone regulation of splanchnic blood vessels using bile duct ligated (BDL) portal hypertensive rats and an in vitro organ culture method. In BDL cirrhosis, the number of ED2-positive resident macrophages increased by two- to fourfold in the vascular walls of the mesenteric artery and extrahepatic portal vein compared with those in sham-operated rats. Many ED1-positive monocytes were also recruited into this area. The expression of inducible nitric oxide (NO) synthase (iNOS) mRNA was increased in the vascular tissues isolated from BDL rats, and accordingly, nitrate/nitrite production was increased. Immunohistochemistry revealed that iNOS was largely expressed in ED1-positive and ED2-positive cells. We further analyzed the effect of iNOS expression on vascular smooth muscle contraction using an in vitro organ culture system. iNOS mRNA expression and nitrate production significantly increased in vascular tissues (without endothelium) incubated with 1 μg/ml lipopolysaccharide (LPS) for 6 h. Immunohistochemistry indicated that iNOS was largely expressed in ED2-positive resident macrophages. α-Adrenergic-stimulated contractility of the mesenteric artery was greatly suppressed by LPS treatment and was restored by NG-nitro-l-arginine methyl ester (NO synthase inhibitor); in contrast, portal vein contractility was largely unaffected by LPS. Sodium nitroprusside (NO donor) and 8-bromo-cGMP showed greater contractile inhibition in the mesenteric artery than in the portal vein with decreasing myosin light chain phosphorylation. In the presence of an α-adrenergic agonist, the mesenteric artery cytosolic Ca2+ level was greatly reduced by sodium nitroprusside; however, the portal vein Ca2+ level was largely unaffected. These results suggest that the induction of iNOS in monocytes/macrophages contributes to a hypercirculatory state in the cirrhosis model rat in which the imbalance of the responsiveness of visceral vascular walls to NO (mesenteric artery >> portal vein) may account for the increased portal venous flow in portal hypertension.

SUMMARYAn isotope dilution method using [1-13C]leucine (Leu) infusion together with open-circuit calorimetry was applied to determine the effect of metabolizable protein (MP) intake on rates of plasma Leu turnover and whole body protein synthesis (WBPS) in six heifers. WBPS rate was estimated from rate of plasma Leu turnover and Leu oxidation to carbon dioxide. The experiment consisted of three levels of MP intake and was conducted in a two 3×3 Latin square designs of three 21-day periods. The experimental diet consisted of mixed hay, maize and soybean meal. Dietary MP intake of each dietary treatment was 4·3, 4·5 and 4·9 g/kg BW0·75/day by changing maize and soybean meal weights. Metabolizable energy (ME) intake was similar for all dietary treatments. When plasma α-[1-13C]keto-isocaproic acid enrichments were used as markers indicating intracellular Leu enrichments, plasma Leu turnover rate (LeuTR) increased (P=0·012) and WBPS tended to increase (P=0·091) as MP intake increased. In contrast, plasma LeuTR and WBPS were not influenced if plasma [1-13C]Leu was taken to indicate intracellular Leu enrichments. Total and plasma Leu oxidation rates did not change but intracellular Leu oxidation increased (P=0·044) with increasing MP intake. In heifers, it is suggested that rates of plasma Leu turnover and WBPS are influenced by dietary MP intake, independent of ME intake, although the change in MP intake was relatively small.

Sano, H., Kajita, M. and Fujita, T. 2007. Effect of dietary protein intake on turnover and oxidation rates of plasma glucose in adult sheep. J. Appl. Anim. Res., 31: 177–180. An isotope dilution method of [U-13C]glucose with open circuit calorimetry was applied to determine the effect of dietary crude protein (CP) intake 5.6, 7.7 and 10.8 g/kg BW0.75 Id on turnover and oxidation rates of plasma glucose in four adult sheep. Isotopic enrichments of plasma [U-13C]glucose and exhaled 13CO2 were determined for calculating the parameters of plasma glucose metabolism. Turnover and oxidation rates of plasma glucose did not change with dietary CP intake levels. It is likely that in adult sheep, plasma glucose metabolism is not influenced by dietary CP intake, when sufficient digestible energy is supplied.

A glucose clamp technique was used to compare dietary starch (ST), starch plus sucrose (ST+SU) and sucrose (SU) with regard to the effect on tissue responsiveness and sensitivity to insulin in intact adult male goats. The goats were fed diets containing 1.2 times of ME and CP for maintenance requirements twice daily for 21 d. Of the energy intake, 30% was offered with ST, ST+SU or SU for the respective diets, and 70% as alfalfa hay, ground corn and ground soybean meal at the respective weight ratio of 1, 1, and 0.3 for all diets. Tissue responsiveness and sensitivity to insulin were evaluated using a hyperinsulinemic euglycemic clamp technique with four levels of insulin infusion beyond 13 h after feeding. The concentrations of plasma metabolites and insulin were also determined at 3, 6 and 13 h after feeding to evaluate the effects of different carbohydrates on metabolic states in the body. Plasma glucose concentration was higher (p = 0.01) for SU diet than for ST and ST+SU diets. Increasing SU intake decreased (p

The responses of whole body protein and glucose kinetics and of nitrogen (N) metabolism to non-protein energy intake (NPEI) were determined using an isotope dilution approach and measurement of N balance in three adult male goats. The diets containing 1.0, 1.5 and 2.0 times ME maintenance requirement, with fixed intake of CP (1.5 times maintenance) and percentage of hay (33%), were fed twice daily for each 21 d experimental period. After an adaptation period of 11 d, N balance was determined over 3 d. On day 17, whole body protein synthesis (WBPS) and glucose irreversible loss rate (ILR) were determined during the absorptive state by a primed-continuous infusion of []phenylalanine, []tyrosine, []tyrosine and []glucose, with simultaneous measurements of plasma concentrations of metabolites and insulin. Ruminal characteristics were also measured at 6 h after feeding over 3 d. Nitrogen retention tended to increase (p) that of tyrosine. Whole body protein synthesis increased (p). These results suggest that increasing NPEI may enhance WBPS and glucose turnover at the absorptive state and improve the efficiency of digestible N retention in goats, with possibly decreased ammonia and increased amino acid absorption. In addition, simultaneous increases in WBPS and glucose ILR suggest stimulatory effect of glucose availability on WBPS, especially when sufficient amino acid is supplied.

A glucose clamp technique was used to investigate the effects of non-protein energy intake on tissue responsiveness and sensitivity to insulin for glucose metabolism in intact adults male goats. Three goats were fed diets at 1.0, 1.5 and 2.0 times of ME for maintenance, each for 21 d. Crude protein intake was 1.5 times of maintenance requirement in each treatment. Tissue responsiveness and sensitivity to insulin were evaluated using a hyperinsulinemic euglycemic clamp technique with four levels of insulin infusion, beginning at 13 h after feeding. Concentrations of plasma metabolites and insulin were also measured at 3, 6 and 13 h after feeding, for evaluating effects of non-protein energy intake on the metabolic status of the animals. Increasing non-protein energy intake prevented an increase in plasma NEFA concentration at 13 h after feeding (p = 0.03). Plasma urea-nitrogen and total amino-nitrogen concentrations decreased (p

Combined experiments of an isotope dilution method of [1- 13 C]leucine with open circuit calorimetry and a nitrogen (N) balance test were applied to determine the effect of dietary crude protein (CP) intake on plasma leucine flux and protein synthesis and degradation in four sheep. The experiment was conducted in a 3×4 Latin rectangle design of three 3-week periods. Dietary CP intake was 5.6, 7.7, and 10.8 g/(kg 0.75 ×d). Metabolizable energy intake was 120% of requirement for all dietary treatments. [1- 13 C]Leucine was intravenously infused for 8 h and blood and breath samples were collected during the latter 2-h period of infusion. Isotopic enrichments of plasma [1- 13 C]leucine, α-[1- 13 C]ketoisocaproic acid, and exhaled 13 CO 2 were determined. For the N balance test, N digestibility, N excretion in urine, and protein balance (N×6.25) increased with increasing dietary CP intake. Rates of plasma leucine turnover, protein synthesis, and degradation changed toward reduction with increased dietary CP intake. It is likely that in sheep, high CP intake enhances protein deposition with reduced protein degradation rather than increased protein synthesis.

Snail and Slug are closely related transcriptional repressors involved in embryonic patterning during metazoan development. In human cancer, aberrant expression of Snail and/or Slug has been correlated with invasive growth potential, a property primarily attributed to their ability to directly repress transcription of genes whose products are involved in cell-cell adhesion, such as E-cadherin, occludin, and claudins. To investigate the molecular mechanisms of alterations in epithelial cell fate mediated by aberrant expression of Snail or Slug, we analyzed the consequences of exogenous expression of these factors in human cancer cells. Aberrant expression of either Snail or Slug led to changes in cell morphology, the loss of normal cell-cell contacts, and the acquisition of invasive growth properties. Snail or Slug expression also promoted resistance to programmed cell death elicited by DNA damage. Detailed molecular analysis revealed direct transcriptional repression of multiple factors with well-documented roles in programmed cell death. Depletion of endogenous Snail by RNA interference led to increased sensitivity to DNA damage accompanied by increased expression of the proapoptotic factors identified as targets of Snail. Thus, aberrant expression of Snail or Slug may promote tumorigenesis through increased resistance to programmed cell death.

The vertebrate Mi-2/NuRD complex is a multi-subunit protein complex containing both histone deacetylase and nucleosome-dependent ATPase subunits. Current models predict that this complex functions primarily in transcriptional repression. Surprisingly, every subunit of this complex presents heterogeneity at the protein and gene level. This raises the intriguing possibility of functional specialization resulting from incorporation of unique gene products into the complex. The MTA (metastasis-associated) proteins represent one class of alternative subunits of the human Mi-2/NuRD complex. The members of this family in human cells are differentially expressed depending on cell type and on physiologic parameters. We summarize evidence supporting the view that the alternative subunits of the complex that have arisen during vertebrate evolution endow unique functional properties.

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) has been believed a key enzyme in tumor invasion, because it is expressed in a variety of malignant human tumors, and overexpression of the enzyme enhances the ability of cellular invasiveness. However, it has not necessarily been clarified whether the endogenously expressed MT1-MMP in human tumors plays a critical role in their invasiveness. We used RNA silencing technology to downregulate the endogenous MT1-MMP expression in human tumor cells (fibrosarcoma HT1080 and gastric carcinoma MKN-28 cell lines), and evaluated the effect on the invasion of a reconstituted basement membrane (Matrigel). Transfection of a double-stranded RNA targeted to the MT1-MMP gene decreased the level of the enzyme to less than 10-20% without affecting production of other MMPs. According to the degree of silencing, activation of proMMP-2 was inhibited. CD44 shedding was also inhibited, but only in part. Decreased MT1-MMP levels were also reflected in reduced cell motility on hyaluronan (HA) and invasion in Matrigel. Thus, specific downregulation of MT1-MMP expression was sufficient to cause significant inhibition of the migration and invasion of tumor cells, even though other MMPs continued to be expressed.

Intact adult male goats were fed diets at 1.0, 1.5 and 2.0 times the metabolizable energy for maintenance, each for 21 days. Supplemental energy above maintenance was supplied as corn starch. Tissue responsiveness and sensitivity to insulin were evaluated using a hyperinsulinemic euglycemic clamp technique with four levels of insulin infusion. The concentrations of plasma metabolites and insulin were also measured at 6h after feeding. At 6h after feeding, plasma total amino-nitrogen, acetate and propionate concentrations were increased (P

Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs have about a 20-amino acid cytoplasmic tail following the transmembrane domain. In contrast, a putative transmembrane domain of MT4-MMP locates at the very C-terminal end, and the expected cytoplasmic tail is very short or nonexistent. Such sequences often act as a glycosylphosphatidylinositol (GPI) anchoring signal rather than as a transmembrane domain. We thus examined the possibility that MT4-MMP is a GPI-anchored proteinase. Our results showed that [(3)H]ethanolamine, which can be incorporated into the GPI unit, specifically labeled the MT4-MMP C-terminal end in a sequence-dependent manner. In addition, phosphatidylinositol-specific phospholipase C treatment released the MT4-MMP from the surface of transfected cells. These results indicate that MT4-MMP is the first GPI-anchored proteinase in the MMP family. During cultivation of the transfected cells, MT4-MMP appeared to be shed from the cell surface by the action of an endogenous metalloproteinase. GPI anchoring of MT4-MMP on the cell surface indicates a unique biological function and character for this proteinase.

The intestinal absorption of an intact oligopeptide was investigated in rats using a synthetic cationic peptide, 001-C8 (H-MeTyr-Arg-MeArg- d -Leu-NH(CH 2 ) 8 NH 2 ). The peptide was coupled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) to prepare a fluorescence-labeled derivative 001-C8-NBD (H-MeTyr-Arg-MeArg- d -Leu-NH(CH 2 ) 8 NH-NBD) for the purpose of quantification. The degradation half-life of 001-C8-NBD in jejunal homogenate (1 mg/mL) was 99.5 min, which was significantly longer than that of natural leucine enkephalin (1.14 min). The absorption of 001-C8-NBD was evaluated by the vascular-perfusion method. Intact 001-C8-NBD appeared in the blood time-dependently and the absorption volume at 30 min (2.75 ± 0.14 μL/cm intestine) was significantly larger than that of [ 3 H]PEG 900 (0.88 ± 0.13 μL/cm intestine), of which membrane permeability is very low. The absorption of 001-C8-NBD was greatly reduced by an adsorptive-mediated endocytosis inhibitor, protamine (10 mM). No inhibition of the absorption of [ 3 H]PEG 900 by protamine was observed. The intestinal absorption was also measured by an in vivo loop method. The absorption clearance of 001-C8-NBD measured by this method (0.083 ± 0.008 μL/min/cm intestine) was comparable to that obtained by the vascular perfusion method (0.092 ± 0.005 μL/min/cm intestine). All of these data suggested that 001-C8-NBD was absorbed as the intact oligopeptide in the intestine in vivo. Adsorptive-mediated transcytosis is suggested to have enormous potential as an oral delivery system for peptide and/or protein drugs.

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.

Aberrant promoter methylation of genes is a common molecular event in breast cancer. Thus, DNA methylation analysis is expected to be a new tool for cancer diagnosis. In this article, we have established a new, high-performance DNA methylation assay, the one-step methylation-specific polymerase chain reaction (OS-MSP) assay, which is optimized for analyzing gene methylation in serum DNA. The OS-MSP assay is designed to detect aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 genes in serum DNA. Moreover, two quality control markers were designed for monitoring the bisulfite conversion efficiency and measuring the DNA content in the serum. Serum samples were collected from patients with primary (n = 101, stages I–III) and metastatic breast cancers (n = 58) as well as from healthy controls (n = 87). If methylation of at least one of the three genes was observed, the OS-MSP assay was considered positive. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24 vs. 8%) and II (26 vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18 vs. 19%) and metastatic breast cancers (55 vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. In conclusion, we have developed a new OS-MSP assay with improved sensitivity and convenience; thus, this assay is more suitable for detecting aberrant promoter methylation in serum DNA. Moreover, the combination of the OS-MSP assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer.

The estrogen receptor (ER)-α (ESR1) is a key regulatory molecule in mammary epithelial cell development. Loss of ER-α in breast cancer is correlated with poor prognosis, increased recurrence after treatment, and an elevated incidence of metastasis. A proposed molecular pathway by which ER-α acts to constrain invasive growth in breast cancer cells involves direct, ER-α-dependent expression of metastasis-associated protein 3, a cell-type-specific component of the Mi-2/NuRD chromatin remodeling complex. MTA3 in turn represses expression of Snail, a transcription factor linked to epithelial to mesenchymal transition and cancer metastasis. To elucidate its role(s) in epithelial to mesenchymal transition (EMT), we expressed Snail in the noninvasive, ER-α-positive MCF-7 cell line. Snail expression led to decreased cell-cell adhesion and increased cell invasiveness. Furthermore, we observed loss of ER-α expression at both the RNA and protein level that was accompanied by direct interaction of Snail with regulatory DNA sequences at the ESR1 locus. A consequence of loss of ER-α function in this system was the increased abundance of key components of the TGF-β signaling pathway. Thus, cross-talk among ER-α, Snail, and the TGF-β pathway appears to control critical phenotypic properties of breast cancer cells.

Responses of whole body protein synthesis (WBPS) and glucose irreversible loss rate (ILR) were compared between dietary starch and sucrose in four male goats. Diets were fed at 1.2 times maintenance requirements of ME and CP with 30% of the ME as starch, starch plus sucrose or sucrose, twice daily. The diets consisted of 33, 32, 11 and 24% of alfalfa hay, corn, soybean meal and the carbohydrates, respectively. The WBPS and glucose ILR during 5 – 7 h after feeding were determined by an isotope dilution method of [2H5]phenylalanine, [2H2]tyrosine, [2H4]tyrosine and [13C6]glucose. Sucrose elevated ammonia nitrogen and lowered acetate concentrations in the rumen, but did not differ from starch in nitrogen retention. Glucose ILR and WBPS were similar between the carbohydrates. It was concluded that dietary sucrose would have effects similar to starch on WBPS and glucose kinetics in the absorptive state in goats fed a high-concentrate diet.

An isotope dilution experiment was conducted to determine the effect of metabolizable energy intake (MEI) as starch on whole body protein synthesis (WBPS), nitrogen (N) retention and glucose irreversible loss rate (ILR) in four adult goats (Capra hircus). The goats were fed isonitrogenous diets containing three different metabolizable energy (1.0, 1.5 and 2.0 times maintenance) twice daily. Energy above maintenance was supplemented with cornstarch. The WBPS and glucose ILR during 5 to 7 h after feeding were measured by a primed-continuous infusion of [2H5]phenylalanine, [2H2]tyrosine, [2H4]tyrosine and [13C6]glucose for 4 h, with measurements of plasma concentrations of metabolites and insulin. Ruminal characteristics were also determined. Increasing MEI improved N retention, despite decreased digestible N. Increasing MEI decreased ruminal pH and ammonia nitrogen. In plasma, decreased urea N, increased total amino N and tyrosine, and trends for increases in phenylalanine and insulin resulted from increasing MEI. Increasing MEI increased ILR of glucose, phenylalanine and tyrosine, and hydroxylation rate of phenylalanine and WBPS. We conclude that in goats increasing MEI as starch enhances WBPS in the absorptive state and N retention, despite a decrease in digestible N. These changes are probably associated with both decreased ammonia absorption and increased amino acid absorption.

Metastasis-associated protein 3 (MTA3) is a cell type-specific subunit of the Mi-2/NuRD transcriptional corepressor complex. In breast cancer cells, MTA3 and the Mi-2/NuRD complex mediate repression of Snail, a transcription factor that promotes epithelial to mesenchymal transitions. Thus, MTA3 functions to maintain a differentiated, epithelial status in breast cancer. Interestingly, in mammary epithelial cells, MTA3 biosynthesis requires both functional estrogen receptor (ER) and estradiol. Here we have investigated the molecular basis for estrogen and ER-dependent expression of MTA3 in breast cancer cells. Molecular dissection of the MTA3 promoter using transient transfection assays identified a composite element required for high-level transcription consisting of an SP1 site in close proximity to a consensus estrogen response element half-site. Depletion of either SP1 or ER-alpha by RNA interference led to loss of MTA3 transcript in multiple breast cancer cell lines, indicating a requirement for both transcription factors in expression of endogenous MTA3. The MTA3 gene thus joins a growing list of loci regulated by both SP1 and ER.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that performs processing of cell surface proteins and degradation of extracellular matrix (ECM) components. Through these proteolytic events, MT1-MMP regulates various cellular functions, including ECM turnover, promotion of cell migration and invasion, and morphogenic responses to extracellular stimuli. MT1-MMP has to be regulated strictly to accomplish its function appropriately at various steps, including at the transcriptional and post-translational levels. MT1-MMP was originally identified as an invasion-promoting enzyme expressed in malignant tumour cells, and also as a specific activator of proMMP-2, which is believed to play a role in invasion of the basement membrane. Since then, it has attracted attention as a membrane-associated MMP that promotes cancer cell invasion and angiogenesis by endothelial cells. Although MT1-MMP has now become one of the best characterized enzymes in the MMP family, there remain numerous unanswered questions. In this chapter, we summarize our recent findings on how MT1-MMP is regulated during cell migration, and how cell migration is regulated by MT1-MMP.

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.

Migratory cells including invasive tumor cells frequently express CD44, a major receptor for hyaluronan and membrane-type 1 matrix metalloproteinase (MT1-MMP) that degrades extracellular matrix at the pericellular region. In this study, we demonstrate that MT1-MMP acts as a processing enzyme for CD44H, releasing it into the medium as a soluble 70-kD fragment. Furthermore, this processing event stimulates cell motility; however, expression of either CD44H or MT1-MMP alone did not stimulate cell motility. Coexpression of MT1-MMP and mutant CD44H lacking the MT1-MMP–processing site did not result in shedding and did not promote cell migration, suggesting that the processing of CD44H by MT1-MMP is critical in the migratory stimulation. Moreover, expression of the mutant CD44H inhibited the cell migration promoted by CD44H and MT1-MMP in a dominant-negative manner. The pancreatic tumor cell line, MIA PaCa-2, was found to shed the 70-kD CD44H fragment in a MT1-MMP–dependent manner. Expression of the mutant CD44H in the cells as well as MMP inhibitor treatment effectively inhibited the migration, suggesting that MIA PaCa-2 cells indeed use the CD44H and MT1-MMP as migratory devices. These findings revealed a novel interaction of the two molecules that have each been implicated in tumor cell migration and invasion.

Five distinct membrane-type matrix metalloproteinases (MT-MMP) have been reported by cDNA cloning. However, the mt4-mmp gene product (MMP-17) has not been identified yet in spite of the cDNA isolation [Puente et al. (1996), Cancer Res. 56, 944–949]. In this study, we re-examined the transcripts for human mt4-mmp by 5′ RACE and identified two types of transcripts. The minor one corresponded to the cDNA reported by Puente et al. and failed to express protein, and the other is the major transcript that has an extended open reading frame and expressed 67 and 71 kDa translation products. Thus, functional mt4-mmp has been identified for the first time.

The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-d-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of125I-001-C8 (7.24 ± 0.41 μl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 μM), protamine (1 mM), poly-l-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-l-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of125I-001-C8 were 1.13 ± 0.23 pmol/mg protein, 0.47 ± 0.43 μM, and 3.13 ± 0.19 μl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8–4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.