[Pathologic analysis of primary cardiac inflammatory myofibroblastic tumor].

OBJECTIVE: To study the clinical and pathologic features of primary cardiac inflammatory myofibroblastic tumor. METHODS: A total of 4 patients with primary cardiac inflammatory myofibroblastic tumor were encountered during the period from 1993 to 2013 in National Center for Cardiovascular Disease. The clinical features, imaging findings and outcomes of the 4 patients were evaluated. ALK protein expression and ALK gene status were studied using the archival tumor tissues. RESULTS: There were 1 female and 3 male patients. The age of patients ranged from 5 months to 30 years (mean = 16 years). The tumor was located in right ventricle (n = 2), right atrium (n = 1) or pericardium (n = 1). Histologic patterns included 2 cases of fibrous histiocytoma type, 1 case of granulomatous type and 1 case of sclerosing type. Immunohistochemical study showed that 2 cases expressed ALK protein. Fluorescence in-situ hybridization however did not reveal any ALK gene rearrangement. CONCLUSIONS: Inflammatory myofibroblastic tumor of the heart is rarely encountered and easily misdiagnosed. It carries distinctive clinical and pathologic features. ALK protein expression is helpful in arriving at the correct diagnosis.

Expression of MMP-2, MMP-9 and MMP-11 in dermatofibroma and dermatofibrosarcoma protuberans.

Dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) are the spindle cell mesenchymal neoplasms of the dermis and subcutis. Their histogenesis still remains uncertain and controversial. Traditionally, CD34 and factor XIIIa or other markers have been widely used to distinguish these two diseases. However, the results of these markers reveal overlapping and they lack specificity. Formalin-fixed, paraffin-embedded blocks were collected from the biopsied cases in Kaohsiung Medical University Hospital in Taiwan between 2004 and 2006. This study included 19 cases of DF and 17 cases of DFSP. Immunohistochemical analysis using antibodies CD34, matrix metalloproteinases (MMP)-2, MMP-9, and MMP-11 was performed. We found that the expression of CD34, MMP-2 and MMP-11 shows significant statistical differences in Immunohistochemistry (IHC) study positive or negative reactivity (positive of CD34 in DFSP and positive of MMP-2 and MMP-11 in DF; p=0.03, p<0.001, and p<0.001, respectively) between DF and DFSP. The result for expression of MMP-9 reveals no differences. The results indicate that the pathogenesis of DF and DFSP are affected by different expressions of extracellular matrix proteins. Metalloproteinases may play a direct role in these two diseases. Since no single marker can completely distinguish DF from DFSP, a combination of more than two or three stains may elevate the accuracy of diagnosis.

Cyclooxygenase-2 expression in dermatofibroma and dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) occasionally resemble each other histologically but differ in histogenesis and biological behavior. This study sought to determine if these lesions can be differentiated by the quantity or quality of expression of cyclooxygenase-2 (COX-2), an enzyme associated with both reactive and neoplastic processes. PATIENTS AND METHODS: Formalin-fixed and paraffin-embedded samples from 20 DFs and 20 DFSPs were stained immunohistochemically with antibodies directed against COX-2. Staining was evaluated semiquantitatively for percentage and intensity using a three-tiered system. DFs were graded and analyzed by cellularity. Findings within the tumors were compared with fibrocyte staining in adjacent tissue. The results were analyzed. RESULTS: Nineteen DFs (95%) and 15 DFSPs (75%) were immunopositive for COX-2; this difference was not statistically significant. Highly cellular DFs showed more widespread (p = 0.0039; r = 0.614) and more intense (p = 0.0586; r = 0.429) staining than less cellular DFs and more prominent staining in adjacent fibroblasts (p = 0.044; r = 0.608). CONCLUSIONS: COX-2 immunostaining does not distinguish DFs from DFSPs. However, the enzyme is expressed more widely and more intensely in more cellular, possibly younger, DFs. The prominent expression of COX-2 in DFSP may have clinical implications for treatment with COX-2 inhibitors in tumors that are not amenable to surgery.

Role of cathepsin K in the turnover of the dermal extracellular matrix during scar formation.

Cathepsins are a group of cysteine proteinases that are involved in various aspects of extracellular matrix turnover. The collagenolytic activity of cathepsin K plays a pivotal role in bone resorption and lung matrix homeostasis, but so far has not been described in skin. To study the role of cathepsin K in the turnover of the cutaneous extracellular matrix, we studied the expression of cathepsin K in human skin and in cultured primary neonatal skin fibroblasts. Normal skin exhibited only low levels or no expression of cathepsin K. In contrast, dermal fibroblasts in surgical scars showed strong cytoplasmic cathepsin K expression. Cathepsin K expression was most prominent in young scars and declined with time. Cultured neonatal primary fibroblasts showed strong cathepsin K staining in the perinuclear endosomal compartment, consistent with intracellular degradation of internalized collagen in lysosomes. Cathepsin K was also found to be strongly expressed in keloids and dermatofibromas, but not in sclerotic areas of morphea. Our data suggest that cathepsin K may play an important role in the homeostasis of the dermal extracellular matrix and the dynamic equilibrium between matrix synthesis and proteolytic degradation, by counteracting deposition of matrix proteins during scar formation with its matrix-degrading activity.

Overexpression of phosphorylated-STAT3 and phosphorylated-ERK protein in dermatofibrosarcoma protuberans.

The overexpression of phosphorylated signal transducer and activator of transcription-3 (p-STAT3) and phosphorylated extracellular signal-regulated kinase (p-ERK) have recently been shown to play an important role in the pathogenesis of various human tumors. However, the role of these two major signal transduction pathways in dermatofibrosarcoma protuberans (DFSP) remains unknown. This study was designed to investigate the significance of p-STAT3 and p-ERK expression in DFSP. The expressions of p-STAT3 and p-ERK were analyzed by immunohistochemical staining in formalin-fixed, paraffin-embedded tissue sections of human DFSP and dermatofibroma. Ten cases were positive for p-STAT3 expression in 14 cases of DFSP, however, only 5 cases were positive in 20 cases of dermatofibroma. Eleven out of 14 cases of DFSP expressed p-ERK, but only four cases were positive in 20 cases of dermatofibroma. The expressions of p-STAT3 and p-ERK were significantly higher than those in dermatofibroma (both p < 0.01). This study suggests that the overexpression of p-STAT3 and p-ERK may play a pivotal role in the oncogenesis of DFSP.

Postradiation malignant fibrous histiocytoma and osteosarcoma of a patient with high telomerase activities.

BACKGROUND: An extremely rare case of postradiation malignant fibrous histiocytoma (MFH) and osteosarcoma (OS) secondary to radiation therapy for leukemia-related osteolytic lesions is presented. In addition, the telomere biology of these tumors was investigated. CASE REPORT: A 14-year-old boy was diagnosed with acute lymphocytic leukemia. The right tibia was irradiated at a total dose of 60 Gy, and the left tibia was irradiated at a total dose of 40 Gy. The left tibia developed MFH and the right tibia developed OS. RESULTS: Telomere reduction (MFH 70.2, OS 70.0%) and high telomerase activities (MFH 12.1, OS 17.7 TPG) were observed. These results reflect an aggressive feature of postradiation sarcomas. CONCLUSION: Prognosis for patients diagnosed with postradiation sarcoma is poor due to its aggressiveness. However, even if sarcoma occurs after irradiation in more than two fields in a single patient, improvements in prognosis are anticipated with appropriate chemotherapies and wide resection.

Inhibitory effect of STI571 on cell proliferation of human malignant fibrous histiocytoma cell lines.

BACKGROUND: Malignant fibrous histiocytoma (MFH) is one of the most common high-grade sarcomas in bone and soft tissue and, due to its chemo-resistance, the prognosis of the disease is poor. ST1571 is a tyrosine kinase inhibitor that was initially developed as a BCR/ABL inhibitor for chronic myeloid leukemia patients. STI571 also selectively inhibits platelet-derived growth factor receptors (PDGFRs) and c-kit. We examined the expression of PDGFRs and c-kit in human MFH cell lines, and the effect of STI571 on cell proliferation. MATERIALS AND METHODS: Four human MFH cell lines (TNMY1, GBS-1, Nara-F and Nara-H) were used. mRNA expression of the receptor tyrosine kinases (PDGFRs and c-kit) was analyzed using reverse transcription-polymerase chain reaction, and the inhibitory effect of STI571 on cell proliferation was analyzed using the MTS assay technique. RESULTS: PDGFRalpha mRNA was expressed in TNMY1 and GBS-1, and PDGFRbeta and c-kit mRNAs were expressed in TNMY1, GBS-1 and Nara-F. All three of these mRNAs were absent in Nara-H. STI571 inhibited cell proliferation of TNMY1, GBS-1 and Nara-F in a dose- and time-dependent manner, but cell proliferation of Nara-H was not inhibited by ST1571 at concentrations of 10 microM or less. CONCLUSION: STI571 significantly inhibited proliferation of the three human MFH cell lines that expressed mRNAs of target receptor tyrosine kinases. The inhibitory effect of ST1571 on cell proliferation in these three cell lines might be due to decreased tyrosine kinase activity. STI571 might be a potent chemotherapeutic agent for human MFHs.

Immunohistochemical expression of matrix metalloproteinases 1, 2, 9, and 14 in dermatofibrosarcoma protuberans and common fibrous histiocytoma (dermatofibroma).

CONTEXT: Common fibrous histiocytoma (cFH) or dermatofibroma and dermatofibrosarcoma protuberans (DFSP) are 2 spindle cell mesenchymal tumors that are distinguished in part by their microscopic growth patterns and clinically by the greater propensity for DFSP to recur. Matrix metalloproteinases (MMPs) potentially play a role in modulating the growth patterns of cFH and DFSP by remodeling the extracellular matrix. OBJECTIVE: To evaluate the immunohistochemical (IHC) expression of MMP-1, MMP-2, MMP-9, and MMP-14 in DFSP and cFH, because (1) MMP-1, MMP-2, MMP-9, and MMP-14 are synthesized by dermal fibroblasts, the major constituent of DFSP and cFH; and (2) platelet-derived growth factor B, which is overexpressed in most examples of DFSP because of t(17;22), activates ets-1, a transcription factor that regulates molecules associated with tumor invasion and metastasis, including MMP-1, MMP-3, and MMP-9. DESIGN: Immunohistochemical studies were performed on archived, formalin-fixed, paraffin-embedded tissue of DFSP (n = 48) and cFH (n = 47).Results.-Significant IHC expression (>10% of tumor cells) in cFH included MMP-14 (27 [59%] of 46 tumors positive), MMP-2 (21 [47%] of 45 tumors positive), MMP-9 (9 [20%] of 45 tumors positive), and MMP-1 (6 [13%] of 46 tumors positive). No DFSPs showed significant IHC expression of any of the MMPs evaluated. However, anti- MMP-2 highlighted a rich microvascular element within deep tumor tissue present in 81% of DFSPs with a prominent subcutaneous component. CONCLUSION: Our IHC results indicate that MMP-1 and MMP-9 are not up-regulated in DFSP. Convincing expression of MMP-14 in cFH suggests that this MMP may affect the growth pattern of the lesion, perhaps by activating MMP-2 expression in tumor cells. In DFSP, MMP-2 may play a role in tumor angiogenesis.

ASK1 (MAP3K5) as a potential therapeutic target in malignant fibrous histiocytomas with 12q14-q15 and 6q23 amplifications.

Malignant fibrous histiocytomas (MFHs) are aggressive tumors without any definable line of differentiation. We recently demonstrated that about 20% of them are characterized by high-level amplifications of the 12q14-q15 chromosome region, associated with either 1p32 or 6q23 band amplification. This genetic finding, very similar to that in well-differentiated liposarcomas, strongly suggests that these tumors actually correspond to undifferentiated liposarcomas. It also suggests that the lack of differentiation could be the consequence of amplification of target genes localized in the 1p32 or 6q23 bands. We report here the characterization by array CGH of the 6q23 minimal region of amplification. Our findings demonstrate that amplification and overexpression of ASK1 (MAP3K5), a gene localized in the 6q23 band and encoding a mitogen-activated protein kinase kinase kinase of the JNK-MAPK signaling pathway, could inhibit the adipocytic differentiation process of the tumor cells. Treatment of a cell line with specific inhibitors of ASK1 protein resulted in the bypass of the differentiation block and induction of a strong adipocytic differentiation. These observations indicate that ASK1 is a target for new therapeutic management of these aggressive tumors.

A selective cyclooxygenase-2 inhibitor, NS-398, inhibits cell growth by cell cycle arrest in a human malignant fibrous histiocytoma cell line.

BACKGROUND: NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor, has been shown to suppress cell proliferation and induce apoptosis in a variety of human cancer cell lines in vitro, except for sarcoma cell lines. The aim of this study was to examine the effect of NS-398 on two human malignant fibrous histiocytoma (MFH) cell lines: MFH-ino and MFH-ToE. MATERIALS AND METHODS: We investigated the effect of NS-398 at various concentrations on the growth of MFH cell lines using cell proliferation assay, cell cycle analysis and EIA assay of PGE2 production. RESULTS: Western blot analysis revealed COX-2 protein expression in both MFH cell lines. NS-398 at 10 or 100 microM resulted in inhibition of the cell proliferation in a dose- and time-dependent manner. NS-398 at 100 microM also induced G0/G1 arrest accompanied by up-regulation of P21WAF1 in MFH-ino cells in a time-dependent manner until 72 hours. NS-398 slightly increased the number of cells in the G2/M-phase and decreased the number in the G0/G1-phase in MFH-ToE cells lacking p53 gene function. Moreover, NS-398 down-regulated PGE2 production in the MFH-ToE cells in a time-dependent manner, but not in the MFH-ino cells. CONCLUSION: Our results indicate that NS-398 inhibits the cell growth and induces G0/G1 arrest in MFH-ino cells accompanied with up-regulation of P21WAF1.

Reactive oxygen-induced carcinogenesis causes hypermethylation of p16(Ink4a) and activation of MAP kinase.

BACKGROUND: Implantation of foreign materials into mice and humans has been noted to result in the appearance of soft tissue sarcomas at the site of implantation. These materials include metal replacement joints and Dacron vascular grafts. In addition, occupational exposure to nickel has been shown to result in an increased risk of carcinogenesis. The molecular mechanisms of foreign body-induced carcinogenesis are not fully understood. MATERIALS AND METHODS: In order to gain insight into these mechanisms, we implanted nickel sulfide into wild type C57BL/6 mice as well as a mouse heterozygous for the tumor suppressor gene, p53. Malignant fibrous histiocytomas arose in all mice, and we have characterized the profile of tumor suppressor genes and signal transduction pathways altered in these cells. RESULTS: All tumors demonstrated hypermethylation of the tumor suppressor gene p16, as well as activation of the mitogen activated protein kinase (MAP kinase) signaling pathway. This knowledge may be beneficial in the prevention and treatment of tumors caused by foreign body implantation. CONCLUSIONS: Oxidative stress induced by nickel sulfide appears to cause loss of p16 and activation of MAP kinase signaling. These findings support the hypothesis of synergistic interactions between MAP kinase activation and p16 loss in carcinogenesis.

Immunohistochemical study of membrane type-matrix metalloproteinases (MT-MMPs) and matrix metalloproteinase-2 (MMP-2) in dermatofibroma and malignant fibrous histiocytoma.

Matrix metalloproteinases (MMPs) play an important role in tumor invasion and metastasis. Enhanced expression of matrix metalloproteinase-2 (MMP-2) has been demonstrated in dermatofibroma (DF) and malignant fibrous histiocytoma (MFH). MMP-2 has been shown to be activated by membrane-type MMPs (MT-MMPs). To study the role of MT-MMP in the activation of MMP-2, skin specimens of DF (five cases) and MFH (three cases) were immunohistochemically studied using in situ zymography and the antibodies against matrix metalloproteinase-2 (MMP-2) and membrane type 1-3-MMPs (MT1-3-MMPs). Both MMP-2 activity and its expression were significantly activated in the tumor cells in DF and MFH. Anti-MT2-MMP strongly reacted with tumor cells of all cases of DF and MFH, whereas anti-MT1 or 3-MMP antibody showed a weak reaction in some cases of DF and MFH. Double immunofluorescence labeling demonstrated that the immunoreactive cells with anti-MMP-2 antibody in DF and MFH consistently reacted with anti-MT2-MMP antibody. The results suggest that the activation of MMP-2 in the benign and malignant fibrous tumors is related to the activation of MT-MMPs.

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma.

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.

Macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

With future exploration of macrophage properties in mind, we established a novel cell line (HS-P) from a transplantable histiocytic sarcoma, derived originally from a tumour in an aged F344 rat. HS-P was subjected to 70 serial passages, in which the mean doubling time was 15.7 h. The cells, which were round, oval or polygonal in shape, were arranged in a compact sheet. They reacted to varying degrees for lysosomal enzymes (acid phosphatase and non-specific esterase) and with the following antibodies: ED1/ED2 (rat macrophage/histiocyte-specific), OX6 (rat MHC class II-specific), lysozyme antibody and alpha1-antichymotrypsin antibody. Electron microscopically, HS-P cells showed lysosomes and prominent cell projections. These findings indicated that the cultured cells were macrophage-like. Syngeneic rats inoculated subcutaneously or intraperitoneally with HS-P cells invariably developed sarcomatous tumours consisting of monomorphic mononuclear cells, which exhibited cytochemical properties similar to those of cultured HS-P cells. Bioassay and reverse transcription-polymerase chain reaction methods revealed that tumour necrosis factor-alpha increased on addition of lipopolysaccharide (LPS), indicating that HS-P cells remained LPS-responsive. HS-P cells may prove to be a useful tool for in-vitro studies of macrophage function.

Expression of telomerase activity and telomerase RNA in human soft tissue sarcomas.

OBJECTIVE: To investigate whether telomerase is reactivated in soft tissue tumor and whether telomerase activity is regulated at the transcriptional level. DESIGN: Fresh tissue samples of 24 soft tissue sarcomas were analyzed for telomerase activity by a radioactive polymerase chain reaction-based telomeric repeat amplification protocol assay and for human telomerase RNA (hTR) by an in situ hybridization assay. SETTING: Tertiary care teaching hospital. PATIENTS: Twenty-four patients with soft tissue tumor were surgically treated. Twelve patients had malignant fibrous histiocytoma, 5 had liposarcoma, 6 had leiomyosarcoma, and 1 had rhabdomyosarcoma. RESULTS: Telomerase activity was detected in 4 sarcoma samples (17%), all of which were positive for hTR. Expression of hTR was demonstrated in 13 sarcomas (54%), 4 of which were positive for telomerase and 9 of which were negative for telomerase. One (50%) of 2 grade 1 tumors, 9 (50%) of 18 grade 2 tumors, and 3 (75%) of 4 grade 3 tumors showed hTR expression. CONCLUSIONS: The relatively low frequency of telomerase activity in soft tissue sarcomas suggests that telomerase may not play an important role in tumorigenesis in these tumors. Telomerase ladders were demonstrated only in association with tumors expressing hTR. It is noteworthy that half of the patients with grade 1 and 2 tumors expressed hTR, suggesting that telomerase RNA may be useful as a marker for identifying tumor aggressiveness earlier than the conventional histopathologic grading scale.

Expression of membrane type 1 matrix metalloproteinase, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 in human cartilaginous tumors with special emphasis on mesenchymal and dedifferentiated chondrosarcoma.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as an activator of the proenzyme of matrix metalloproteinase 2 (MMP-2: gelatinase A), and has also been shown to play a crucial role in tumor invasion by activating proMMP2 in both lung and gastric carcinoma. The tissue inhibitor of metalloproteinase 2 (TIMP-2) plus the MT1-MMP complex also plays an important role in the activation of proMMP-2. In this study, the expressions of MT1-MMP, MMP-2 and TIMP-2 were evaluated in 10 enchondromas, 34 conventional chondrosarcomas, 5 clear-cell chondrosarcomas, 7 mesenchymal chondrosarcomas and 8 dedifferentiated chondrosarcomas. The expressions were immunohistochemically visualized on paraffin sections and the levels of expression were assessed semiquantitatively. The extent of staining was assessed by the extent score in order to determine the overall level of expression. The extent scores of MT1-MMP, MMP-2 and TIMP-2 in grade 2 chondrosarcoma were significantly higher than those in either enchondroma or grade 1 chondrosarcoma (P < 0.05). In conventional chondrosarcoma, significant correlations were found between the extent scores of MT1-MMP and MMP-2 (P < 0.001), MT1-MMP and TIMP-2 (P < 0.01), and MMP-2 and TIMP-2 (P < 0.01). The undifferentiated small round tumor cells of mesenchymal chondrosarcoma showed lower positive rates and extent scores for MT1-MMP (2/7, 0.7 +/- 0.5) and MMP-2 (3/7, 0.7 +/- 0.4) than for cartilaginous components of mesenchymal chondrosarcoma [MT1-MMP (4/7, 1.3 +/- 0.5) and MMP-2 (7/7, 1.9 +/- 0.3)] or conventional chondrosarcoma. In dedifferentiated chondrosarcoma, the extent scores of MT1-MMP, MMP-2 and TIMP-2 in low-grade cartilaginous components were not significantly different from those in conventional chondrosarcoma; however, the high-grade anaplastic components showed high extent scores for MT1-MMP, MMP-2 and TIMP-2, compared with the low-grade cartilaginous components of dedifferentiated chondrosarcoma or conventional chondrosarcoma. According to our results, the expression of MT1-MMP as well as that of MMP-2 or TIMP-2 demonstrated a significant correlation with the tumor grade in human cartilaginous tumors. Furthermore, the expressions of MT1-MMP, MMP-2 and TIMP-2 were also found to play a crucial role in invasion in the high-grade components of dedifferentiated chondrosarcoma.

Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.

We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mast cytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcAbeta1-R alpha1,4-N -acetylgalactosaminyltransferase. This is the first identification of an alpha1,4-N-acetylgalactosaminyltransferase. Using N -acetylchondrosine GlcAbeta1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-( N -morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N -acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 microM, 188 microM, and 27 microM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcAbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcbeta1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O -sulfate)beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cbeta1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.

Expression of cathepsins in dermal fibrous tumors: an immunohistochemical study.

Cathepsins are lysosomal proteases that are distributed in many normal tissues and are primarily responsible for intracellular catabolism and turnover. The increased level of cathepsins in tumors together with their ability to degrade extracellular matrix proteins has led to the hypothesis that they are involved in the process of invasion and metastasis. We studied immunohistochemically the expression of cathepsins B, pro-D and pro-L in 8 cases of dermatofibrosarcoma protuberans (DFS), five cases of atypical fibroxanthoma (AFX) and twenty cases of dermatofibroma (DF). Expression of cathepsins B and pro-D could be detected in 5 of the 8 cases (62.5%) of DFS, whereas cathepsin pro-L was found in 4 (50%) cases. All AFX expressed cathepsin pro-L, whereas cathepsins B and pro-D were observed in 4 out of 5 cases. None of the malignant tumors showed a recurrence or metastasis after a period of four years. We found no expression of cathepsins in DF. In the epidermis and appendages, an expression of cathepsins pro-D, pro-L and B was seen. We conclude that cathepsins may be markers of increased metabolism rather than specific markers of malignancy.

Caspase-mediated apoptosis in AK-5 tumor cells: a cell-free study using peptide inhibitors and antisense strategy.

An in vitro system has been employed to study the apoptotic mechanisms in the AK-5 tumor which is a spontaneously regressing rat histiocytoma. Cytosolic extracts of tumor cells primed for apoptosis using dexamethasone and immune serum from tumor-regressing animals were able to induce apoptosis in intact nuclei and reproduce the classical morphological and biochemical features typical of apoptotic cells. The cleavage of lamin A and PARP to signature fragments by these extracts and the inhibition of the same using peptide inhibitors signify the pivotal role of ICE and ICE-related proteases in apoptosis. Lamin A cleavage was insensitive to YVAD but PARP cleavage was blocked by both YVAD and DEVD. Cell extracts derived from cells overexpressing the Bcl-2 gene and Nedd-2 antisense gene, respectively, failed to induce apoptosis in exogenously added nuclei, suggesting that Bcl-2 gene product is downregulating a key event in apoptotic cascade. The study also demonstrates the coherent action of different ICE-related proteases in apoptosis and their functional redundancy. This system may prove useful for analyzing complex molecular mechanisms underlying apoptosis in tumor cells.