Ag-catalysed cutting of multi-walled carbon nanotubes.

In this work, the cutting of carbon nanotubes is investigated using silver nanoparticles deposited on arc discharge multi-walled carbon nanotubes. The composite is subsequently heated in air to fabricate shortened multi-walled nanotubes. Complementary transmission electron microscopy and spectroscopy techniques shed light on the cutting mechanism. The nanotube cutting is catalysed by the fundamental mechanism based on the coordination of the silver atoms to the π-bonds of carbon nanotubes. As a result of the metal coordination, the strength of the carbon-carbon bond is reduced, promoting the oxidation of carbon at lower temperature when heated in air, or lowering the activation energy required for the removal of carbon atoms by electron beam irradiation, assuring in both cases the cutting of the nanotubes.

Outcome determinants for transformed indolent lymphomas treated with or without autologous stem-cell transplantation.

BACKGROUND: Transformation of indolent lymphomas (IL) to an aggressive histology (TIL) often results in a rapid clinical course, treatment refractoriness and shortened survival. Although rituximab-containing regimens (R-chemo) have become standard of care in CD20-positive TIL, the role of autologous stem-cell transplantation (ASCT) is still debated. The purpose of this study was to determine whether the outcome of TIL patients improved if they, at transformation, also received ASCT. Furthermore, we investigated the outcome of cases with histologically low- and high-grade components diagnosed either simultaneously or after a period of overt indolent disease. We also analyzed, whether prior rituximab treatment during the indolent course of the disease affected outcome after transformation. PATIENTS AND METHODS: Eighty-five patients (≤68 years) with histologically confirmed TIL were included. Five-year overall (OS) and progression-free survival (PFS) were calculated. Selected parameters were tested in a multivariate analysis. All analyses were conducted on three cohorts: (i) whole cohort (all TIL), (ii) patients with co-existing evidence of both indolent and aggressive histology at diagnosis (Composite/discordant TIL) and (iii) patients transformed after prolonged prior indolent disease (sequential TIL). RESULTS: Fifty-four patients (64%) received ASCT consolidation and 31 (36%) did not. Within the 'all TIL' cohort, the 5-year OS and PFS for R-chemo + ASCT versus R-chemo alone, were 67% versus 48% (P = 0.11) and 60% versus 30% (P = 0.02), respectively. Furthermore, in 'Composite/discordant TIL' R-chemo + ASCT showed no impact on OS (76% versus 67%; P = 0.66) or PFS (71% versus 62%; P = 0.54). Conversely, R-chemo + ASCT improved the outcome of 'sequential TIL' (OS 62% versus 36%; P = 0.07; PFS 53% versus 6%; P = 0.002), regardless of prior rituximab therapy. The beneficial effect of ASCT was significantly higher in patients who had not received rituximab at IL stage. CONCLUSIONS: ASCT improved the outcome in sequential, but not composite/discordant TIL. The beneficial impact of ASCT was greater in patients, who were rituximab-naïve at transformation.

Beta Cell Regenerating Potential of Azadirachta indica (Neem) Extract in Diabetic Rats.

OBJECTIVE: This study evaluated the ability of 0.8% neem leaf extract (NLE) to treat diabetes mellitus by assessing its effects on blood glucose, insulin levels and islet morphology in streptozotocin (STZ)-induced diabetic Sprague-Dawley rats. METHODS: Diabetes was induced in two to three-day old rat pups by STZ intraperitoneally (60 mg/kg), followed by a further 40 mg/kg dose 12-23 weeks later. The diabetic treated (DT) rats received 0.8% w/v NLE in tap water while diabetic control (DC) and normal control (NC) rats received water ad libitum. Body weight, water and chow consumption, and blood glucose were evaluated weekly. Blood and pancreas were collected at the end of the study to evaluate serum insulin and islet histology, respectively. RESULTS: Neem leaf extract (0.8%) improved weight gain and beta cell regeneration but did not reduce blood glucose. Serum insulin increased slightly in the treated group and three-fold in the DC group (p < 0.05). CONCLUSION: The results suggest that NLE has beta cell regenerating potential.

R-CHOEP-14 improves overall survival in young high-risk patients with diffuse large B-cell lymphoma compared with R-CHOP-14. A population-based investigation from the Danish Lymphoma Group.

BACKGROUND: Optimal treatment of young patients with high-risk diffuse large B-cell lymphoma (DLBCL) remains a matter of debate and requires improvement. The combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP) has in other patient groups been shown to be effective. Further improvement has been accomplished with the use of rituximab in combination with the regimens every 2 weeks (R-CHOP-14, R-CHOEP-14). The aim of the present retrospective population-based study was to compare R-CHOP-14 with R-CHOEP-14 in a cohort of high-risk patients aged 18-60 years with two or more risk factors (stage III-IV, elevated lactate dehydrogenase levels, performance status 2-4). To our knowledge, this is the first study comparing these two regimens in this patient group. METHODS: We obtained data for the period 2004-2009 from the Danish Lymphoma Database. One hundred and fifty-nine patients were eligible to enter the study. Primary end point was overall survival (OS) and secondary end points were response to treatment, progression-free survival (PFS) and safety. RESULTS: Four-year OS was superior in the R-CHOEP-14 group: 75% compared with 62% for R-CHOP-14 (P=0.04). This superiority was also seen for PFS: 4-year PFS was 70% for the R-CHOEP-14 group compared with 58% for the R-CHOP-14 group (P=0.02). CONCLUSION: R-CHOEP-14 is a promising regimen for young patients with high-risk DLBCL with improved OS and PFS compared with R-CHOP-14.

A dual action of saturated fatty acids on electrical activity in rat pancreatic ß-cells. Role of volume-regulated anion channel and KATP channel currents.

Free fatty acids (FFAs) exert complex actions on pancreatic ß-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of ß-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic ß-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyper-polarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic ß-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic ß-cell function.

Comparison of antibiotic resistance and virulence factors in pigmented and non-pigmented Pseudomonas aeruginosa.

OBJECTIVE: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However pigment production appeared to be more significantly associated with multi-drug resistance, presence of virulence-associated genes, and expression of certain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status of an isolate.

The emergence of qnr-mediated quinolone resistance among Enterobacteriaceae in Jamaica.

OBJECTIVE: Quinolone resistance is usually caused by various chromosomal mutations, but has been more recently associated with plasmids which carry the qnr determinant. The aim of this study is to investigate the prevalence of qnr genes in clinical isolates of Enterobacteriaceae in Jamaica. METHODS: A total of 255 non-duplicate fluoroquinolone-resistant Enterobacteriaceae clinical isolates, comprising 232 Escherichia coli, 20 Klebsiella species and three Enterobacter spp were collected between October 2007 and November 2008 from hospitalized patients in Jamaica. The presence of the qnr gene was screened by PCR using specific primers for qnrA, qnrB and qnrS in extracted plasmid DNA. RESULTS: Eighty-three (32.5%) of these isolates were qnr-positive, of which 47.0% housed the qnrA gene only, 1.2% qnrB and 9.6% qnrS only. Another 36.1% possessed both qnrA and qnrS genes. Approximately 30% of the quinolone-resistant E coli isolates harboured the qnr gene while 50% Klebsiella spp and all Enterobacter spp were positive. CONCLUSION: The emergence of qnr-mediated quinolone resistance among clinical Enterobacteriaceae isolates is described for the first time in Jamaica.

Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression.

The differentiated phenotype of rabbit articular chondrocytes was modulated in primary culture by treatment with 1 microgram/ml retinoic acid (RA) and reexpressed in secondary culture by treatment with the microfilament-disruptive drug dihydrocytochalasin B (DHCB) in the absence of RA. Because the effective dose of DHCB (3 microM) did not elicit detectable cell rounding or retraction, the nature and extent of microfilament modification responsible for induction of reexpression was evaluated. The network of microfilament stress fibers detected with rhodamine-labeled phalloidin in primary control chondrocytes was altered by RA to a "cobblestone" pattern of circularly oriented fibers at the cell periphery. Subsequent treatment with DHCB resulted in rapid changes in this pattern before overt reexpression. Stress fibers decreased in number and were reoriented. Parallel arrays of long fibers that traversed the cell were evident, in addition to fiber fragments and focal condensations of staining. Immunofluorescent staining of intermediate filaments revealed a marked decrease in complexity and intensity during RA treatment but no change during reexpression. An extended microtubular architecture was present throughout the study. These results clearly identify microfilaments as the principal affected cytoskeletal element and demonstrate that their modification, rather than complete disruption, is sufficient for reexpression. The specificity of DHCB and the reorientation of these filaments before reexpression of the differentiated phenotype suggests a causative role in the mechanism of reexpression.

Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape.

Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA-modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture.

An appraisal of ultramicrotomy, FIBSEM and cryogenic FIBSEM techniques for the sectioning of biological cells on titanium substrates for TEM investigation.

Ultramicrotomy, focused ion beam scanning electron microscopy (FIBSEM) and cryogenic FIBSEM (cryo-FIBSEM) techniques, as developed for the controlled cross-sectioning of mesenchymal stem cells (MSCs) and human osteoblasts (HObs) on titanium (Ti) substrates for transmission electron microscopy (TEM) investigation, are compared. Conventional ultramicrotomy has been used to section cells on Ti-foil substrates embedded in resin, but significant problems with cell detachment using this technique restricted its general applicability. Conventional FIBSEM 'lift-out' procedures were found to be effective for the preparation of uniform sections of fixed and dehydrated cell/Ti specimens, but the control of cell staining remains an issue. Cryo-FIBSEM procedures used with an 'H-bar' sample geometry enabled the sectioning of fixed and hydrated cell/Ti specimens, but issues remain over ion beam-induced artefacts and control of frost on the sample foils.

KCC3 and KCC4 expression in rat adult forebrain.

Potassium chloride ion cotransporters (KCCs) are part of a family of transporters classically described as being involved in cell volume regulation. Recently, KCC2 has been shown to have a role in the development of the inhibitory actions of amine transmitters, whereas KCC3 also plays a fundamental role in the development and function of the central and peripheral nervous system. We have re-assessed the expression of each of the known KCCs in the rat forebrain using RT-PCR and in situ hybridisation histochemistry. As well as confirming the widespread expression of KCC1 and KCC2 throughout the brain, we now show a more restricted expression of KCC3a in the hippocampus, choroid plexus and piriform cortex, as well as KCC4 in the choroid plexus and the suprachiasmatic nucleus of the hypothalamus. The expression of KCC4 in the latter and KCC2 in the lateral hypothalamic and ventromedial hypothalamic nuclei suggests that these cotransporters may have selective roles in neuroendocrine or homeostatic functions. Finally, we demonstrate the existence of a truncated splice variation of KCC3a in the rat that appears to be expressed exclusively in neurons (as is KCC2), whereas the native form of KCC3a and KCC4 appears to be expressed in glial cells.

Association between expression of activated 72-kilodalton gelatinase and tumor spread in non-small-cell lung carcinoma.

BACKGROUND: Matrix metalloproteinases, in particular the 92-kd and 72-kd gelatinases, have been implicated in the progression of breast, colorectal, and gastric carcinomas, but involvement of the gelatinases in progression of non-small-cell lung carcinoma has not been documented. Immunohistochemical studies have measured the overall expression of these enzymes in tumor tissue but have failed to determine the proportion of active enzyme to latent proenzyme. Because the conversion of the latent proenzyme to active enzyme results in removal of a 10-kd amino-terminal domain, the expression of each proteinase can be determined by zymography, which separates substances according to molecular weight. PURPOSE: The purpose of this study was to examine the expression and activation of 92-kd and 72-kd proenzymes in non-small-cell lung carcinoma. METHODS: Gelatin zymography was used to study the expression of 92-kd and 72-kd gelatinases in 22 samples of non-small-cell lung carcinoma and adjacent uninvolved tissue. Medium conditioned by human RPMI-7951 melanoma cells was used as a marker for the 72-kd proenzyme, and medium conditioned by concanavalin A-treated human HT-1080 fibrosarcoma cells was used as a marker for both the 92-kd proenzyme and the 62-kd activated form of the 72-kd proenzyme. RESULTS: Both 92-kd and 72-kd proenzymes were expressed to varying degrees in the samples studied. The 82-kd activated form of the 92-kd proenzyme was detected in eight tumor samples but in none of the matched uninvolved tissues. Expression of the 62-kd activated form of the 72-kd proenzyme ranged from a strong band in the tumor tissue, with little or none detectable in the adjacent uninvolved tissue, to the presence of only trace amounts of enzyme in both tumor and uninvolved tissue. There was, however, a highly significant statistical association between the level of expression of the 62-kd activated enzyme in the tumor tissue and evidence of tumor spread (P = .001). CONCLUSION: These results demonstrate elevated expression of the activated forms of both the 92-kd and 72-kd proenzymes in non-small-cell lung carcinoma tissue relative to adjacent uninvolved tissue. IMPLICATION: These results indicate that non-small-cell lung carcinoma should be considered as a possible target for metalloproteinase inhibitory therapy.

Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases.

BACKGROUND: The importance of matrix metalloproteinases in angiogenesis, tumor growth, and metastasis is well known. However, little is known about the role of matrix metalloproteinases in the formation of hemangiomas and about the possible therapeutic use of matrix metalloproteinase inhibitors in aggressive vascular tumors. PURPOSE: To study the role of matrix metalloproteinase in vascular tumors, we tested the antineoplastic activity of a synthetic inhibitor of matrix metalloproteinases, batimastat, on an experimental model of hemangioma, formed by murine endothelioma cells transformed by polyoma middle-T oncogene (eEnd.1). METHODS: The effect of batimastat was studied in vivo on the formation of hemorrhaging, cavernous hemangiomas by eEnd.1 endothelioma cells injected subcutaneously in nude mice and on the angiogenic response induced by an endothelioma cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel). The effect of batimastat was investigated in vitro on endothelial cell proliferation, motility, and invasion of a layer of Matrigel. RESULTS: Daily treatment with batimastat (30, 3, and 0.3 mg/kg at the site of eEnd.1 cell injection) inhibited tumor growth, with increased doubling time. The carboxamide derivative of batimastat, BB-374, a poor inhibitor of matrix metalloproteinase activity, was less active in reducing hemangioma growth. Histologic analysis of treated tumors indicated a reduction in the size of blood-filled spaces and in hemorrhage. Batimastat also inhibited the angiogenic response induced by cultured eEnd.1 endothelioma cell supernatant embedded in a pellet of Matrigel. Batimastat significantly inhibited endothelial cell invasion in vitro through a layer of Matrigel, but it showed no direct cytotoxic activity. CONCLUSIONS: Batimastat reduces in vivo growth of experimental hemangiomas, most probably by blocking endothelial cell recruitment by the transformed cells or by interfering with cell organization in vascular structures. IMPLICATIONS: These results confirm the importance of matrix metalloproteinase in endothelial cell recruitment that occurs in angiogenesis and in the formation of vascular tumors and suggest a therapeutic potential for synthetic matrix metalloproteinase inhibitors.

Tumor cell invasion inhibited by TIMP-2.

The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.

Matrix metalloproteinase inhibitor BB-94 (batimastat) inhibits human colon tumor growth and spread in a patient-like orthotopic model in nude mice.

Matrix metalloproteinases have been implicated in the growth and spread of metastatic tumors. This role was investigated in an orthotopic transplant model of human colon cancer in nude mice using the matrix metalloproteinase inhibitor BB-94 (batimastat). Fragments of human colon carcinoma (1-1.5 mm) were surgically implanted orthotopically on the colon in 40 athymic nu/nu mice. Administration of BB-94 or vehicle (phosphate buffered saline, pH 7.4, containing 0.01% Tween 80) commenced 7 days after tumor implantation (20 animals/group). Animals received 30 mg/kg BB-94 i.p. once daily for the first 60 days and then 3 times weekly. Treatment with BB-94 caused a reduction in the median weight of the primary tumor from 293 mg in the control group to 144 mg in the BB-94 treated group (P < 0.001). BB-94 treatment also reduced the incidence of local and regional invasion, from 12 of 18 mice in the control group (67%) to 7 of 20 mice in the treated group (35%). Six mice in the control group were also found to have metastases in the liver, lung, peritoneum, abdominal wall, or local lymph nodes. Only two mice in the BB-94 group had evidence of metastatic disease, in both cases confined to the abdominal wall. The reduction in tumor progression observed in the BB-94-treated group translated into an improvement in the survival of this group, from a median survival time of 110 days in the control group to a median survival time of 140 days in the treated group (P < 0.01). Treatment with BB-94 was not associated with any obvious toxic effect, and these results suggest that such agents may be effective as adjunctive cancer therapies.

A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts.

We have examined the effect of a synthetic low-molecular-weight matrix metalloproteinase inhibitor, [4-(N-hydroxyamino)-2R-isobutyl-3S- (thiopen-2-ylthiomethyl)-succinyl]-L-phenylalanine-N-meth yla mide (BB-94), on human ovarian carcinoma xenografts growing in nude mice. The xenografts grew as thick intraperitoneal mucinous ascites containing free-floating tumor cell clumps. The ascites increased in volume, causing death approximately 3 weeks after introduction. Treatment with BB-94 caused resolution of ascitic disease. Tumor burden was dramatically reduced, and survival increased 5-6-fold. The increase in survival was dose dependent. The effects observed with BB-94 appeared to be due to its matrix metalloproteinase inhibiting effects, inasmuch as its inactive diastereoisomer had no effect on tumor biology. Following treatment with BB-94, free-floating clumps of tumor cells became surrounded by a capsule of host cells. These clumps of tumor cells typically formed one small (approximately 8 mm) avascular tumor of bright white appearance loosely attached to fat in the peritoneum. Tumor cells within these capsules often appeared to be necrotic. Gel substrate analysis demonstrated that activated Mr 92,000 type IV collagenase was present in the xenografts. We propose that inhibition of this enzyme causes the transition of ascites to solid tumors, concomitantly slowing tumor cell growth and allowing the development of tumor stroma.

Inhibition of organ invasion by the matrix metalloproteinase inhibitor batimastat (BB-94) in two human colon carcinoma metastasis models.

The effect of the matrix metalloproteinase inhibitor batimastat was evaluated in two human colorectal cancer metastasis models involving: (a) the liver-invasive tumor C170HM2 and (b) the lung-invasive tumor AP5LV, both of which have been shown to express the M(r) 72,000 type IV collagenase. Batimastat at concentrations between 0.01 and 3.0 micrograms/ml had no direct cytotoxic effects on the in vitro growth of the cell lines. In the liver-invasive tumor model, batimastat administered i.p. from day 10 to termination of the therapy (day 39) at 40 mg/kg reduced both the mean number of liver tumors (35% of vehicle-treated control; P < 0.05) and the cross-sectional area of the tumors (43% of vehicle-treated control; P < 0.05). In the lung-invasive tumor model, batimastat administered daily (40 mg/kg i.p.) significantly reduced tumor weight within the lung (72% of vehicle-treated control; P < 0.05) but did not significantly affect nodule number. In the latter model, in which the take rate was unaffected, tumor cells were introduced into the lateral tail vein, and lung localization may have been a physical phenomenon not involving invasion. In the former model, tumor cells were introduced directly into the peritoneal cavity, and from there the cells adhered to and invaded the liver capsule. Because the take rate is significantly reduced, it may be that the matrix metalloproteinases are involved in this process. Batimastat may be a therapeutic modality for the treatment of colorectal cancer metastasis.

Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the matrix metalloproteinase inhibitor batimastat (BB-94).

We examined the effects of the synthetic matrix metalloproteinase inhibitor batimastat (BB-94) on lung colonization and spontaneous metastasis of a rat mammary carcinoma, HOSP.1P. This tumor expresses both latent and active forms of the matrix metalloproteinases MMP-2 and MMP-9, although the former, as in human breast cancer, is the most prominent. Administration of batimastat (6 x 30 mg/kg i.p.) inhibited by up to 80% both the number and median weights of HOSP.1P lung colonies following i.v. inoculation of cells. This implies an effect both on seeding efficiency and subsequent tumor development. In spontaneous metastasis assays, limited treatment with batimastat (commencing when s.c. tumors were established and continuing until 5 or 14 days after their surgical removal) significantly inhibited lung metastasis but had little effect on lymphatic metastasis. However, when treatment was initiated 2 days prior to surgery and continued until day 70, 100% of animals survived to day 120 when there was no evidence of metastatic disease. All control animals (n = 25) in two separate experiments died before day 100 with lymphatic, lung, and extrapulmonary metastases. Taken together, these data suggest that lymphatic dissemination by HOSP.1P tumor cells is less susceptible to inhibition by batimastat than vascular invasion, but that long-term treatment can effectively prevent the outgrowth of putative micrometastases in both lymph nodes and lungs, allowing sustained tumor-free survival.

Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines.

The regulation of Mr 72,000 type IV collagenase and interstitial collagenase expression was studied in vitro. Three tumorigenic human cell lines were used, together with human fetal lung fibroblasts as a nontumorigenic control. Mr 72,000 type IV collagenase was expressed constitutively by all four cell lines, whereas only A2058 melanoma cells exhibited constitutive expression of interstitial collagenase. Treatment of cells with transforming growth factor beta 1 (TGF-beta 1) and 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed an opposite pattern of regulation of these two metalloproteinases. Specifically, TPA increased interstitial collagenase mRNA levels in each cell line and decreased type IV collagenase mRNA levels in control fibroblasts and the tumorigenic cell lines, HT-1080 and A2058. TGF-beta 1 treatment increased type IV collagenase mRNA levels in each cell line and decreased interstitial collagenase mRNA levels in A2058 melanoma cells. Interstitial collagenase mRNA induction was accompanied in all cell lines by elevated interstitial procollagenase in the conditioned medium, as detected by zymography. Changes in Mr 72,000 type IV collagenase expression revealed a more complex pattern of regulation. TPA and TGF-beta 1 treatment of HT-1080 cells resulted in the appearance of two bands of gelatinolytic activity with a molecular weight of approximately 62,000 and 59,000. The Mr 62,000 species was also induced by TGF-beta 1 treatment of A2058 cells. Addition of affinity-purified radiolabeled Mr 72,000 type IV procollagenase to TPA-treated HT-1080 cells demonstrated that both species were products of the Mr 72,000 proenzyme and that exogenous proenzyme could be processed by these cells. Western blot analysis with specific antipeptide antibodies revealed that both the Mr 62,000 and 59,000 species were derived from the Mr 72,000 proenzyme by amino-terminal cleavage. There was no evidence for cellular processing of either interstitial procollagenase or the Mr 92,000 type IV procollagenase. These results demonstrate that the Mr 72,000 type IV collagenase is under the control of different regulatory elements from interstitial collagenase, at the level of both mRNA expression and cellular processing, and that this processing appears to be the result of a phorbol ester and TGF-beta 1-inducible cellular activation mechanism. The ratio of active enzyme species to latent Mr 72,000 proenzyme may provide a better correlation with invasive potential than overall levels of this widely expressed metalloproteinase.

Cloning and characterization of human tumor cell interstitial collagenase.

A full-length complementary DNA (cDNA) for interstitial collagenase was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The tumor interstitial collagenase cDNA was sequenced and compared to the published sequences for human fibroblast collagenase. The sequence for the tumor collagenase has two DNA base pairs which differ from the sequence of normal fibroblast collagenase. Restriction enzyme digestion of a specific DNA fragment produced by polymerase chain reaction amplification of genomic DNA from human placenta resolves a discrepancy in the previously reported DNA and amino acid sequences for the fibroblast collagenase. A high level of expression of interstitial collagenase message was found in human A2058 melanoma cells by Northern blot analysis, and this level was slightly increased by phorbol ester (phorbol myristate acetate) stimulation. Interstitial collagenase mRNA expression was significantly decreased by treatment with either transforming growth factor-beta 1 or retinoic acid in A2058 melanoma cells. A high level of the collagenase protein secreted into conditioned media was identified by Western blotting. As shown by gelatin zymogram analysis interstitial collagenase was one of at least two metalloproteinases secreted by this same cell line. Thus, human melanoma cells can directly produce interstitial collagenase without a requirement for host cell interaction.