Malignant transformation and antineoplastic actions of nonsteroidal antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts.

In this study, we use primary embryonic fibroblasts derived from cyclooxygenase-deficient transgenic embryos to further investigate the role of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), in the process of neoplastic transformation. Cells with either, neither, or both of the cyclooxygenases were transformed by Ha-ras and/or SV40. Our results show that when a cyclooxygenase enzyme is present, the transformed cells have marked increases in COX-2 and/or COX-1 expression. Nevertheless, each type of cell, deficient in either or both cyclooxygenases, can be readily transformed at almost equal efficiency. Different nonsteroidal antiinflammatory drugs (NSAIDs) were used to examine their possible antineoplastic effects on the transformed cells, which have various levels of expression of COX-1 or COX-2. Our results show that NSAIDs suppress the colony formation in soft agar in a dosage-dependent manner in the absence of the cyclooxygenase(s). Thymidine incorporation and apoptosis analyses further demonstrate that the NSAIDs are effective in the cyclooxygenase-null cells. Our findings with cyclooxygenase knockout cells confirm recent reports that some of the antiproliferative and antineoplastic effects of NSAIDs are independent of the inhibition of either COX-1 or COX-2. They also show that transformation is independent of the status of cyclooxygenase expression, suggesting that the involvement of the cyclooxygenases in tumorigenesis may occur at later steps.

Compensatory prostaglandin E2 biosynthesis in cyclooxygenase 1 or 2 null cells.

Prostaglandin E2 (PGE2) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1-deficient (COX-1(-/-)) or cyclooxygenase 2-deficient (COX-2(-/-)) mice was examined after treatment with interleukin (IL)-1beta, tumor necrosis factor alpha, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1(-/-) or COX-2(-/-) cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1-induced expression of cytosolic phospholipase A2 (cPLA2), a key enzyme-regulating substrate mobilization for PGE2 biosynthesis, was also more pronounced in both COX-1(-/-) and COX-2(-/-) cells. Thus, COX-1(-/-) and COX-2(-/-) cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents.

Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture.

We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E(2) (PGE(2)) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2(-/-)) compared with wild-type (PGHS-2(+/+)) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D(3) (1,25-D) or by parathyroid hormone (PTH), was reduced by 60-70% in PGHS-2(-/-) cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE(2). Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2(-/-) than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2(-/-) mice. Histological examination of bone from 5-week-old PGHS-2(-/-) mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25-D and PTH.

Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2.

To investigate roles in intestinal inflammation for the 2 cyclooxygenase (COX) isoforms, we determined susceptibility to spontaneous and induced acute colitis in mice lacking either the COX-1 or COX-2 isoform. We treated wild-type, COX-1(-/-), COX-2(-/-), and heterozygous mice with dextran sodium sulfate (DSS) to provoke acute colonic inflammation, and we quantified tissue damage, prostaglandin (PG) E(2), and interleukin-1beta. No spontaneous gastrointestinal inflammation was detected in mice homozygous for either mutation, despite almost undetectable basal intestinal PGE(2) production in COX-1(-/-) mice. Both COX-1(-/-) and COX-2(-/-) mice showed increased susceptibility to a low-dose of DSS that caused mild colonic epithelial injury in wild-type mice. COX-2(-/-) mice were more susceptible than COX-1(-/-) mice, and selective pharmacologic blockade of COX-2 potentiated injury in COX-1(-/-) mice. At a high dose, DSS treatment was fatal to 50% of the animals in each mutant group, but all wild-type mice survived. DSS treatment increased PGE(2) intestinal secretion in all groups except COX-2(-/-) mice. These results demonstrate that COX-1 and COX-2 share a crucial role in the defense of the intestinal mucosa (with inducible COX-2 being perhaps more active during inflammation) and that neither isoform is essential in maintaining mucosal homeostasis in the absence of injurious stimuli.

Targeted disruption of the mouse topoisomerase I gene by camptothecin selection.

Topoisomerase I has ubiquitous roles in important cellular functions such as replication, transcription, and recombination. In order to further characterize this enzyme in vivo, we have used gene targeting to inactivate the mouse Top-1 gene. A selection protocol using the topoisomerase I inhibitor camptothecin facilitated isolation of embryonic stem cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal selection procedures. The disrupted Top-1 allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages. Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expression of topoisomerase I is not required for gamete viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-1 gene was also shown to be linked to the agouti locus.

Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice.

Two isoforms of cyclooxygenase (COX) are known, and to date most studies have implicated COX-2, rather than COX-1, as the isoform involved in colon carcinogenesis. In the present study, we show that homologous disruption of either Ptgs-1 or Ptgs-2 (genes coding for COX-1 or COX-2, respectively) reduced polyp formation in Min/+ mice by approximately 80%. Only COX-1 protein was immunohistochemically detected in normal intestinal tissue, whereas both COX-1 and variable levels of COX-2 protein were detected in polyps. Prostaglandin E2 was increased in polyps compared with normal tissue, and both COX-1 and COX-2 contributed to the PGE2 produced. The results indicate that COX-1, as well as COX-2, plays a key role in intestinal tumorigenesis and that COX-1 may also be a chemotherapeutic target for nonsteroidal anti-inflammatory drugs.

Cyclooxygenase-2 contributes to functional hyperemia in whisker-barrel cortex.

The prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) is expressed in selected cerebral cortical neurons and is involved in synaptic signaling. We sought to determine whether COX-2 participates in the increase in cerebral blood flow produced by synaptic activity in the somatosensory cortex. In anesthetized mice, the vibrissae were stimulated mechanically, and cerebral blood flow was recorded in the contralateral somatosensory cortex by a laser-Doppler probe. We found that the COX-2 inhibitor NS-398 attenuates the increase in somatosensory cortex blood flow produced by vibrissal stimulation. Furthermore, the flow response was impaired in mice lacking the COX-2 gene, whereas the associated increase in whisker-barrel cortex glucose use was not affected. The increases in cerebral blood flow produced by hypercapnia, acetylcholine, or bradykinin were not attenuated by NS-398, nor did they differ between wild-type and COX-2 null mice. The findings provide evidence for a previously unrecognized role of COX-2 in the mechanisms coupling synaptic activity to neocortical blood flow and provide an insight into one of the functions of constitutive COX-2 in the CNS.

Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo-oxygenase 2 (COX-2) and mice that lack either COX-1 (COX-1-/-) or COX-2 (COX-2-/-) to show that COX-2, but not COX-1, plays an important role in wear debris-induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild-type and COX-1-/- mice. COX-2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild-type and COX-1-/- mice, COX-2-/- mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX-2 is an important mediator of wear debris-induced osteolysis and suggests that COX-2 inhibitors are potential therapeutic agents for the prevention of wear debris-induced osteolysis.

Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta.

Mice carrying a null mutation for either of the two cyclooxygenase (COX) isoenzymes, necessary for prostanoid production, exhibit several isotype-specific reproductive abnormalities. Mice deficient in COX-1 are fertile but have decreased pup viability, whereas mice deficient in COX-2 fail to ovulate and have abnormal implantation and decidualization responses. The present study identifies the specific contribution of each COX isoenzyme in hypothalamic, pituitary, and ovarian function and establishes the pathology and rescue of the anovulatory syndrome in the COX-2-deficient mouse. In both COX-1- and COX-2-deficient mice, pituitary gonadotropins were selectively increased, whereas hypothalamic LHRH and serum gonadotropin levels were similar to those in wild-type animals (+/+). No significant differences in serum estrogen or progesterone were noted among the three genotypes. Exogenous gonadotropin stimulation with PMSG and hCG produced a comparable 4-fold increase in ovarian PGE2 levels in wild-type and COX-1(-/-) mice. COX-2(-/-) mice had no increase in PGE2 over PMSG-stimulated levels. Wild-type and COX-1(-/-) mice ovulated in response to PMSG/hCG; very few COX-2(-/-) animals responded to this regimen. The defect in ovulation in COX-2 mutants was attributed to both an abnormal cumulus oophorum expansion and subsequent stigmata formation. Gonadotropin stimulation and concurrent treatment with PGE2 or interleukin-1beta resulted in ovulation of COX-2(-/-) mice comparable to that in COX-2(+/+), whereas treatment with PGF2alpha was less effective. Collectively, these data demonstrate that COX-2, but not COX-1, is required for the gonadotropin induction of ovarian PG levels; that COX-2-related prostanoids are required for stabilization of the cumulus oophorum during ovulation; and that ovulation can be restored in the COX-2(-/-) animals by simultaneous treatment with gonadotropins and PGE2 or interleukin-1beta.

Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1beta.

Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.

The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2(-/-), but not in cyclooxygenase-1(-/-) mice.

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.

Disruption of the mouse cyclooxygenase 1 gene. Characteristics of the mutant and areas of future study.

Surprisingly, disruption of the COX-1 gene resulted in generally healthy mice. This is in spite of the fact that prostaglandin levels in the tissues examined were reduced by greater than 99%. The results obtained to date with the COX-1 deficient mice indicate that some of the physiological roles previously attributed to COX-1 may not be entirely correct. Ongoing studies with the COX deficient mice are aimed at better defining the physiological roles of the cyclooxygenases and concomitantly the mechanisms by which NSAIDs cause their biological effects.

Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I.

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.

Phenotypic selection and characterization of mutant alleles of a eukaryotic DNA topoisomerase I.

We have developed a simple, effective genetic screen for mutant alleles of eukaryotic DNA topoisomerase I that manifest severely depressed or complete loss of enzymatic function. The screen is based on the extreme toxicity of vaccinia topoisomerase expression in the Escherichia coli lysogen strain BL21(DE3) and is notable for its ease in distinguishing nonsense mutations (that result in truncated proteins) from missense mutations. The power of the method is evinced by our observation that 100% of the candidate alleles identified in the screen were ultimately found to have single-base changes at the DNA level that result in amino acid substitutions at the protein level. By mutagenizing plasmid DNA in vitro with hydroxylamine and applying this phenotypic screen, we have isolated five distinct single amino acid substitution mutants, each of which shows a biochemical phenotype, that is, greater than or equal to 90% reduction in specific DNA relaxing activity of the mutant protein relative to wild type. The amino acids thus implicated in topoisomerase function have identical or related counterparts at homologous positions in the topoisomerases from yeast and man. The same genetic screen has been applied to the selection of temperature-sensitive alleles of the vaccinia topoisomerase, leading to the isolation of two additional single-hit mutant alleles that display a temperature-sensitive growth phenotype in E. coli BL21(DE3). By broadening our mutagenesis procedures, we expect to generate a comprehensive map of vaccinia topoisomerase function and primary protein structure that should have direct application to eukaryotic cellular enzymes. Our methodology should be applicable to the selection of missense and conditional mutant alleles in other genes whose expression in bacteria is toxic.

Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli.

The D1 gene encoding the large subunit of vaccinia virus mRNA capping enzyme was expressed in Escherichia coli BL21(DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. Two major species of protein-GMP complex were formed, one of Mr 95,000 (corresponding in size to the D1 gene product) and one of Mr 60,000. Partial purification of the guanylyltransferase was effected by ammonium sulfate precipitation and ion-exchange chromatography. The expressed large subunit synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor, but was unable to catalyze cap methylation in the presence of S-adenosylmethionine. Thus, the small capping enzyme subunit was shown to be dispensable for guanylylation, but required for cap methylation of RNA. The Mr 95,000 and Mr 60,000 protein-GMP forming activities were resolved during centrifugation in a glycerol gradient; the two forms sedimented at 5.5 S and 4.4 S, respectively, consistent with each enzyme form being a monomer. Either species catalyzed GMP transfer to an RNA acceptor. The isolated Mr 95,000 guanylyltransferase could be converted to an active Mr 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the Mr 95,000 polypeptide.

Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice.

BACKGROUND: Genetic ablation of cyclooxygenase-2 (COX-2) resulted in cystic renal dysplasia and early death in adult mice. The ontologic development of the renal pathology and the biochemical and physiological abnormalities associated with the dysplasia are unknown. METHODS: Mice homozygous for a targeted deletion of COX-2 (-/-) were compared with wild-type littermates (+/+). Somatic and kidney growth and renal histology were studied at the day of birth and at a number of postnatal ages. Systolic blood pressure, urinalysis, urine osmolality, serum and urine chemistries, and inulin clearance were evaluated in adult animals. RESULTS: Beginning at postnatal day 10 (PN10), kidney growth was suppressed in -/- animals, while somatic growth and heart growth were unaffected. By PN10, -/- kidneys had thin nephrogenic cortexes and crowded, small, subcapsular glomeruli. The pathology increased with age with progressive outer cortical dysplasia, cystic subcapsular glomeruli, loss of proximal tubular mass, and tubular atrophy and cyst formation. Adult -/- kidneys had profound diffuse tubular cyst formation, outer cortical glomerular hypoplasia and periglomerular fibrosis, inner cortical nephron hypertrophy, and diffuse interstitial fibrosis. The glomerular filtration rate was reduced by more than 50% in -/- animals (6.82 +/- 0.65 mL/min/kg) compared with wild-type controls (14.7 +/- 1.01 mL/min/kg, P < 0. 001). Plasma blood urea nitrogen and creatinine were elevated in null animals compared with controls. Blood pressure, urinalysis, urine osmolality, and other plasma chemistries were unaffected by the deletion of COX-2. CONCLUSIONS: Deficiency of COX-2 results in progressive and specific renal architectural disruption and functional deterioration beginning in the final phases of nephrogenesis. Tissue-specific and time-dependent expression of COX-2 appears necessary for normal postnatal renal development and the maintenance of normal renal architecture and function.

Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.

Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.

Lack of cyclooxygenase-2 inhibits growth of teratocarcinomas in mice.

Two isoforms of cyclooxygenase (COX-1 or COX-2) have been identified in the prostanoid biosynthetic pathway. The constitutive form, COX-1, is thought to maintain cellular homeostasis and the inducible form, COX-2, is recognized as a primary response gene thought to be involved in modulating cell proliferation and differentiation. To further characterize the role of the cyclooxygenases in cell proliferation, differentiation, and tumorigenicity we developed embryonic stem (ES) cell lines which contain homozygous disruptions in either the COX-1 or the COX-2 gene. These lines were then examined in terms of their viability, proliferation, and in vitro differentiation potential. Our results demonstrate that the wild-type ES cells do not express either COX-1 or COX-2 until the cells undergo differentiation. And the lack of either cyclooxygenase has no apparent effect on ES cell proliferation in vitro. However, the absence of a functional COX-2 gene leads to a dramatic reduction in the formation and growth of teratocarcinomas that appear when ES cells are injected into syngeneic mice. Histological microscopy shows that the few very small tumors that were generated from ES cells lacking COX-2 appear more differentiated than tumors emerging from COX-1 -/- or wild-type cells by exhibiting greater keratinization in the areas of squamous epithelium and the ossification of bone-forming cartilage. We conclude that the presence of a functional COX-2 enzyme is necessary for the efficient growth of these teratocarcinomas in animals.

Genetic evidence for distinct roles of COX-1 and COX-2 in the immediate and delayed phases of prostaglandin synthesis in mast cells.

Activation of mast cells by aggregation of their high-affinity IgE receptors stimulates prostaglandin (PG) D(2) synthesis and secretion. An immediate phase of PGD(2) synthesis, complete within 30 min, is followed by a delayed, second phase of PGD(2) production that reaches a maximum 4 to 8 h after activation. Activation of mast cells from COX-2 (-/-) mice stimulates the release of PGD(2) during the first 30 min, whereas activation of mast cells from COX-1 (-/-) mice does not generate any PGD(2) in the first 2 h. On the other hand, COX-2 (-/-) cells do not participate in delayed phase of PGD(2) synthesis, while COX-1 (-/-) cells secrete low levels of PGD(2) between 2 and 4 h after activation. These data demonstrate that (i) the first phase of PG synthesis is COX-1 dependent and (ii) the second, delayed phase of PG synthesis is dependent on activation-induced synthesis and activity of COX-2.