Mechanisms of extracellular S0 globule production and degradation in Chlorobaculumtepidum via dynamic cell-globule interactions.

The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.

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.

Fibrosarcoma cells transduced with the IL-6 gene exhibited reduced tumorigenicity, increased immunogenicity, and decreased metastatic potential.

Murine fibrosarcoma cell lines transduced with retroviral vectors containing the murine interleukin 6 (IL-6) gene constitutively secreted IL-6. When injected s.c. into normal mice these IL-6-secreting tumors exhibited reduced tumorigenicity. This reduced tumorigenicity was not seen in nude or irradiated mice, implicating a T-cell-dependent, radiosensitive host response activated by the cytokine. Subcutaneous IL-6-secreting tumor did not retard the growth of distant deposits of wild-type tumor in the same host. However, animals rejecting IL-6-secreting tumors exhibited resistance to later challenge with wild-type tumor. When injected i.v. in an experimental metastasis model the IL-6-secreting tumors failed to or were extremely inefficient in giving rise to pulmonary nodules; this was observed in both normal and immunoincompetent mice, implicating a second, nonimmune mechanism affecting the growth of the tumor modified to secrete IL-6.

Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinoma.

Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.

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.

Synthesis, characterization, and polymerization activity of [bis(4,4'-bis(neophyldimethylsilylmethyl)-2,2'-bipyridyl)copper(I)]+CuBr2- and Implications for copper(I) catalyst structures in atom transfer radical polymerization.

A series of 4,4'-disilyl-substituted-2,2'-bipyridine ligands were prepared using a metathesis reaction of the dianion of 4,4'-dimethyl-2,2'-bipyridine with several trialkylsilyl chlorides: 4,4'bis(tert-butyldimethylsilylmethyl)-2,2'-bipyridine (dTBDMSbipy), 4,4'-bis(dimethylthexylsilylmethyl)-2,2'-bipyridine (dTHEXbipy), and 4,4'-bis(neophyldimethylsilylmethyl)-2-2'-bipyridine (dNEObipy). It was observed that the side chain length correlated with the ability of the ligand to form hydrocarbon soluble complexes of copper(I) bromide, with dNEObipy forming the most soluble and easily crystallized complexes. The atom transfer radical polymerization (ATRP) of styrene using dNEObipy as the ligand displayed molecular weight control equivalent to other ATRP systems in which solubilizing ligands, such as 4,4'-di-5-nonyl-2,2'-bipyridine or 4,4'-di-n-heptyl-2,2'-bipyridine, were used. The one-to-one complex of dNEObipy with CuBr was prepared and its crystal structure was determined. The resulting complex had the ionic formulation [(dNEObipy)2Cu]+[CuBr2]- and displayed similar activities in styrene ATRP as the standard 2 dNEObipy/CuBr catalyst system. These and other polymerization results in addition to NMR experiments suggest that the predominant copper(I) species formed in ATRP solutions is the 2-to-1 ligand-to-copper(I) cation, [(dNEObipy)2Cu]+, with either a dihalocuprate or halide counteranion, depending upon the conditions.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues.

Human tissue inhibitor of metalloproteinase-2 (TIMP-2) was cloned and sequenced from an A2058 human melanoma cell cDNA library. When the sequence was compared with that of human TIMP-1 at both the nucleotide and deduced amino acid levels, the homology appeared closer at the protein level than at the nucleotide level, suggesting that these inhibitors diverged early in the evolution of this gene family. Comparison of the deduced amino acid sequence for TIMP-2 with that of human TIMP-1 shows that there are two regions in which the similarity is below the overall average of 66%. It is postulated that these regions are responsible for the unique ability of TIMP-2 to bind to the latent form of the 72-kDa type IV collagenase. Polyclonal anti-TIMP-2 antisera recognized TIMP-2 but not TIMP-1 on immunoblotting. Northern blot analysis of RNA from A2058 human melanoma, HT-144 human melanoma, HT-1080 human fibrosarcoma, and WI-38 fetal lung fibroblast cell lines demonstrated two distinct transcripts of 1.0 and 3.5 kilobases (kb) for timp-2 mRNA. Both transcripts are down-regulated in response to transforming growth factor-beta but are unchanged in response to phorbol ester treatment. This is in contrast to the up-regulation of timp-1 transcripts by these agents and indicates that timp-2 and timp-1 are independently regulated in cell culture. Northern blot analyses of matched normal and tumor tissue samples from five cases of human colorectal carcinoma were performed. Normal and tumor tissues contain both the 1.0- and 3.5-kb transcripts. However, in the tissue samples the ratio of the 3.5-kb transcript to the 1.0-kb transcript was markedly elevated. No evidence of down-regulation of timp-2 transcript levels was noted in the tumor tissues. This is in contrast to the elevated timp-1 transcript levels seen in these tumor samples. Thus, timp-2 mRNA transcript levels are differentially regulated from timp-1 levels in vivo as well as in cell culture.

Association of nm23-H1 allelic deletions with distant metastases in colorectal carcinoma.

A prospective study analysed the prognostic value of nm23-H1 allelic deletions in colorectal cancer. Of 21 patients with no evidence of distant metastases at initial operation, 11 showed nm23-H1 allelic deletions (including 1 homozygous deletion); 10 had no nm23-H1 deletions. After median follow-up of 25 months, distant metastases had developed in 8 of 11 (73%) patients with nm23-H1 deletions but in only 2 of 10 (20%) without nm23-H1 deletions (p less than 0.03). Tests with probe YNZ 22.1, near p53, showed no significant association with distant metastases. nm23-H1 may be, or may be located near, a late-acting suppressor gene in colorectal carcinoma, in which deletions may have prognostic value.