Induction of collagenase production in U937 cells by phorbol ester and partial purification of the induced enzyme.

The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons.

Use of lectin affinity chromatography for the purification of collagenase from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNLs) store collagenase in an inactive form in secretory granules. The enzyme can be activated in vitro by limited proteolysis or by sulfhydryl-modifying agents such as N-ethylmaleimide (NEM). We have enriched NEM-activated collagenase 820-fold using granule isolation, gel filtration, and wheat germ agglutinin (WGA)-agarose chromatography. The use of WGA-agarose resulted in a 55-fold enrichment of collagenase in a single step with very little loss of activity. The chromatographic behavior of collagenase on other lectin matrices was explored and gave information about the type of complex asparagine-linked oligosaccharide found on collagenase isolated from PMNLs.

Simian virus 40-infected, interferon-treated cells contain 2',5'-oligoadenylates which do not activate cleavage of RNA.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Preparation, characterization, and use of an antiserum specific for 2',5'-oligoadenylates.

Periodate-oxidized ppp(A2'p)3A was coupled in high yield to an acyl hydrazide derivative of bovine serum albumin and the conjugate was used to immunize rabbits. Several potent and specific antisera were obtained, and one was tested extensively. Fifty per cent of the bound probe ppp(A2'p)3A3'[32P]pCp was displaced by compounds containing the moiety -pA2'pA-at concentrations of 20-60 nM and by compounds containing the moiety -p(A2'p)2A- at 1 nM. The 3',5'-oligoadenylate (A3'p)3A was bound more than 10,000 times less tightly than (A2'p)3A. The antiserum can be used in competition assays in which the amount of an antibody-probe complex is measured after binding to Millipore filters or to polystyrene beads. These assays allow the clear detection of as little as 20 fmol (20 microliters of 1 nM) of 2',5'-oligoadenylates.

Analysis of 2',5'-oligoadenylates in cells and tissues.

Complex mixtures of 2',5'-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2',5'-oligoadenylates. Representative results from three different biological systems are presented. The function of 2',5'-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.