Interleukin-2 receptor-α proximal promoter hypomethylation is associated with multiple sclerosis.

Genetic studies have demonstrated association between single-nucleotide polymorphisms within the IL2RA (interleukin-2 receptor α-subunit) gene and risk of developing multiple sclerosis (MS); however, these variants do not have obvious functional consequences. DNA methylation is a source of genetic variation that could impact on autoimmune disease risk. We investigated DNA methylation of the IL2RA promoter in genomic DNA obtained from peripheral blood mononuclear cells and neural tissue using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A differential methylation profile of IL2RA was identified, suggesting that IL2RA expression was regulated by DNA methylation. We extended our analysis of DNA methylation to peripheral blood mononuclear cell (PBMC) of MS cases and controls using MALDI-TOF and Illumina HumanMethylation450 arrays. Analyses of CpG sites within the proximal promoter of IL2RA in PBMC showed no differences between MS cases and controls despite an increase in IL2RA expression. In contrast, we inferred significant DNA methylation differences specific to particular leukocyte subtypes in MS cases compared with controls by deconvolution of the array data. The decrease in methylation in patients correlated with an increase in IL2RA expression in T cells from MS cases in comparison with controls. Our data suggest that differential methylation of the IL2RA promoter in T cells could be an important pathogenic mechanism in MS.

Microtubules and actin filaments: dynamic targets for cancer chemotherapy.

Microtubules and actin filaments play important roles in mitosis, cell signaling, and motility. Thus these cytoskeletal filaments are the targets of a growing number of anti-cancer drugs. In this review we summarize the current understanding of the mechanisms of these drugs in relation to microtubule and actin filament polymerization and dynamics. In addition, we outline how, by targeting microtubules, drugs inhibit cell proliferation by blocking mitosis at the mitotic checkpoint and inducing apoptosis. The beta-tubulin isotype specificities of new anticancer drugs and the antitumor potential of agents that act on the actin cytoskeleton are also discussed.

Assessing the utility of in vitro microtubule assays for studying mechanisms of peripheral neuropathy with the microtubule inhibitor class of cancer chemotherapy.

The microtubule inhibitor (MTI) class of chemotherapeutics provide an effective treatment for several different types of cancers, however, severe chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting toxicity in patients that limits their use. While CIPN was predicted with MTIs based on histopathology and functional effects in non-clinical toxicology studies, these investigations often require large numbers of animals and long term studies. As in vitro MT assays have been used for decades to study mechanisms of efficacy, we hypothesized that those same assays could be used to study mechanisms of peripheral neuropathy and predict severe CIPN. We analyzed published data on in vitro microtubule (MT) properties for different MTIs that cause varying levels of peripheral neuropathy in patients. Eribulin, vinorelbine and vinfluinine, which all have less severe CIPN than the vinca alkaloids or taxanes, have unique MT properties consisting of reduced affinity and limited binding to MTs (i.e. bind only to the ends and not along the length). Binding more potently to tubulin in the absence of neuronal BIII tubulin was also observed with eribulin and may suggest specificity for tumor tubulin over neuronal tubulin. These are possible mechanisms for causing less severe deleterious effects on MTs in peripheral nerves leading to reduced severity of CIPN. Our analyses demonstrated that in vitro tools used to study the mechanisms of action in inducing severe CIPN (i.e MTI interactions with MTs) warrant further investigation and may be useful for developing next generation MTIs with reduced CIPN.

Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling.

The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.

Historical fragmentation of islands and genetic drift in populations of Galápagos lava lizards (Microlophus albemarlensis complex).

The formation of islands following a rise in sea level at the end of Pleistocene is expected to disrupt the equilibrium between genetic drift and gene flow in species with limited ability to disperse. Here, we test the hypothesis that genetic drift in isolation has caused the differentiation of Galápagos lava lizards (Microlophus albemarlensis complex) found on 12 islets that are likely to have been connected to a larger island, Isla Santa Cruz, during the late Pleistocene. Using 11 microsatellite loci, screened on 524 individuals from 17 localities distributed among and within 15 islands, we found marked differences in allelic richness and heterozygosity. Genetic differentiation was strong (global F(ST) = 0.44), with pairwise differences found among populations on islets being larger than differences among three localities sampled within Isla Santa Cruz. As expected under a scenario of drift in isolation, there was a positive correlation of genetic diversity with island size, no relationship between genetic and geographical distance and a strong negative correlation between heterozygosity and measures of genetic differentiation. We conclude that seawater is a significant barrier to gene flow in lava lizards on this timescale. Our results suggest that the shallow diversification of the M. albemarlensis complex is not due to recent gene flow and that genetic drift may have played a substantial role in observed patterns of phenotypic variation among islands.

A P300-based brain-computer interface for people with amyotrophic lateral sclerosis.

OBJECTIVE: The current study evaluates the efficacy of a P300-based brain-computer interface (BCI) communication device for individuals with advanced ALS. METHODS: Participants attended to one cell of a N x N matrix while the N rows and N columns flashed randomly. Each cell of the matrix contained one character. Every flash of an attended character served as a rare event in an oddball sequence and elicited a P300 response. Classification coefficients derived using a stepwise linear discriminant function were applied to the data after each set of flashes. The character receiving the highest discriminant score was presented as feedback. RESULTS: In Phase I, six participants used a 6 x 6 matrix on 12 separate days with a mean rate of 1.2 selections/min and mean online and offline accuracies of 62% and 82%, respectively. In Phase II, four participants used either a 6 x 6 or a 7 x 7 matrix to produce novel and spontaneous statements with a mean online rate of 2.1 selections/min and online accuracy of 79%. The amplitude and latency of the P300 remained stable over 40 weeks. CONCLUSIONS: Participants could communicate with the P300-based BCI and performance was stable over many months. SIGNIFICANCE: BCIs could provide an alternative communication and control technology in the daily lives of people severely disabled by ALS.

Differential Morphological and Biochemical Recovery from Chemotherapy-Induced Peripheral Neuropathy Following Paclitaxel, Ixabepilone, or Eribulin Treatment in Mouse Sciatic Nerves.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Evaluation of the in situ, time-integrated DGT technique by monitoring changes in heavy metal concentrations in estuarine waters.

Various natural and anthropogenic processes influence heavy metal concentrations within estuaries. In situ, time-integrated DGT measurements made over concurrent tidal phases found significantly higher concentrations of Cu (probability p=0.017), Zn (p=0.003) and Ni (p=0.003) during the flood phase, because the incoming tide passes several point sources. DGT-reactive Cu concentrations significantly decreased with increased tidal-flushing and vice versa within a marina (correlation r=-0.788, p=0.02). DGT measurements also recorded significant increases in Cu (4 out of 4 sites, p<0.001) and Zn (3 out of 4 sites, p< or =0.015) after a 24 mm rainfall event. Finally, DGT-reactive Cu increased significantly (p<0.001) during peak boating times, due to increased numbers of Cu-antifouled boats. This study demonstrates that, with judicious selection of deployment times, DGT measurements enable changes in heavy metal concentrations to be related to various cycles and events within estuaries.

Taxol suppresses dynamics of individual microtubules in living human tumor cells.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.

Comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro.

Vinepidine, a new derivative of vincristine, and three clinically used Catharanthus derivatives, vinblastine, vincristine, and vindesine, were examined for their abilities to inhibit net tubulin addition at the assembly ends of bovine brain microtubules at steady state. Although all four derivatives were generally similar in potency, their relative abilities to inhibit tubulin addition were distinguishable. Vinepidine and vincristine were the most potent derivatives (Ki, 0.079 +/- 0.018 (SD) microM and 0.085 +/- 0.013 microM, respectively), followed by vindesine (Ki, 0.110 +/- 0.007 microM) and vinblastine (Ki, 0.178 +/- 0.025 microM). In contrast to their relative abilities to inhibit microtubule assembly in vitro, vinblastine and its derivative, vindesine, were generally more potent than vincristine and vinepidine in inhibiting cell proliferation in culture. Vinblastine was nine times more potent than the weakest derivative, vinepidine, in B16 melanoma cells. In L-cells, vinblastine completely inhibited growth at 40 nM, whereas vincristine and vindesine caused about 25% inhibition, and vinepidine was inactive. When B16 melanoma cells were treated with drug before being injected into mice, retardation of tumor growth was best achieved with vindesine, one of the weaker of the four derivatives in vitro. The results demonstrate that chemical differences among the Catharanthus derivatives, which affect to small extents the abilities of the derivatives to inhibit microtubule assembly in vitro, result in significant differences in the order and the magnitude of the abilities of the drugs to inhibit cell growth.

Mechanism of inhibition of cell proliferation by Vinca alkaloids.

We have used a structure-activity approach to investigate whether the Vinca alkaloids inhibit cell proliferation primarily by means of their effects on mitotic spindle microtubules or by another mechanism or by a combination of mechanisms. Five Vinca alkaloids were used to investigate the relationship in HeLa cells between inhibition of cell proliferation and blockage of mitosis, alteration of spindle organization, and depolymerization of microtubules. Indirect immunofluorescence staining of microtubules and 4,6-diamidino-2-phenylindole staining of chromatin were used to characterize the effects of the drugs on the distributions of cells in stages of the cell cycle and on the organization of microtubules and chromosomes in metaphase spindles. The microtubule polymer was isolated from cells and quantified using a competitive enzyme-linked immunoadsorbent assay for tubulin. We observed a nearly perfect coincidence between the concentration of each Vinca derivative that inhibited cell proliferation and the concentration that caused 50% accumulation of cells at metaphase, despite the fact that the antiproliferative potencies of the drugs varied over a broad concentration range. Inhibition of cell proliferation and blockage of cells at metaphase at the lowest effective concentrations of all Vinca derivatives occurred with little or no microtubule depolymerization or spindle disorganization. With increasing drug concentrations, the organization of microtubules and chromosomes in arrested mitotic spindles deteriorated in a manner that was common to all five congeners. These results indicate that the antiproliferative activity of the Vinca alkaloids at their lowest effective concentrations in HeLa cells is due to inhibition of mitotic spindle function. The results suggest further that the Vinca alkaloids inhibit cell proliferation by altering the dynamics of tubulin addition and loss at the ends of mitotic spindle microtubules rather than by depolymerizing the microtubules. The specific alterations of spindle microtubule dynamics appear to differ among the five Vinca congeners, and such differences may be responsible for differences in the antitumor specificities of the drugs.

Low potency of taxol at microtubule minus ends: implications for its antimitotic and therapeutic mechanism.

In many cells, low concentrations of Taxol potently block mitosis at the transition from metaphase to anaphase, with no change in microtubule polymer mass and no microtubule bundling. Mitotic block ultimately results in apoptotic cell death and appears to be the most potent antitumor mechanism of Taxol (M. A. Jordan et al., Cancer Res. 56: 816-825, 1996). Mitotic inhibition results, at least in part, from stabilization of growing and shortening dynamics, specifically at the plus ends of microtubules, by the binding of very few Taxol molecules to the microtubule surface (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993; W. B. Derry et al., Biochemistry, 34: 2203-2211, 1995). A number of actions of Taxol on mitotic spindle function may be due to its effects on microtubule dynamics at the minus ends of microtubules, effects that previously have not been described. Here, we determined the effects of Taxol on minus ends of purified microtubules at steady state. In contrast to the strong stabilizing effects on plus ends, substoichiometric ratios of Taxol bound to tubulin in microtubules did not affect growing, shortening, or dynamicity at minus ends. Thus, in blocked mitotic cells, Taxol can potently suppress dynamics at plus ends of spindle microtubules, whereas its impotence at minus ends permits continued microtubule depolymerization at the spindle poles. Differential effects of Taxol at opposite microtubule ends may explain Taxol's actions on spindle structure and function and its unique potent antitumor action.

Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death.

Paclitaxel at low concentrations (10 nM for 20 h) induces approximately 90% mitotic block at the metaphase/anaphase transition in HeLa cells, apparently by suppressing dynamics of spindle microtubules (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993). It is not known, however, whether inhibition of mitosis by such low paclitaxel concentrations results in cell death. In the present work, we found that after removal of paclitaxel (10 nM-1 microM), blocked cells did not resume proliferation. Instead, cells exited mitosis abnormally within 24 h. They did not progress through anaphase or cytokinesis but entered an interphase-like state (chromatin decondensed, and an interphase-like microtubule array and nuclear membranes reformed). Many cells (> or = 55%) contained multiple nuclei. Additional DNA synthesis and polyploidy did not occur. DNA degradation into nucleosome-sized fragments characteristic of apoptosis began during drug incubation and increased after drug removal. Cells died within 48-72 h. Incubation with paclitaxel (10 nM for 20 h) resulted in high intracellular drug accumulation (8.3 microM) and little efflux after paclitaxel removal; intracellular retention of paclitaxel may contribute to its efficacy. The results support the hypothesis that the most potent chemotherapeutic mechanism of paclitaxel is kinetic stabilization of spindle microtubule dynamics.

Effects of Tau and MAP2 on the interaction of maytansine with tubulin: inhibitory effect of maytansine on vinblastine-induced aggregation of tubulin.

Maytansine, a potent inhibitor of mitosis and in vitro microtubule assembly, was used to demonstrate a striking difference in the mechanism by which two of the main groups of brain microtubule-associated proteins, Tau and MAP2, interact with tubulin. At the low concentrations of 0.5 to 2 microM, maytansine inhibited Tau-catalyzed tubulin assembly more effectively than it did MAP2-catalyzed assembly. This effect differed markedly from that of vinblastine, although both drugs bind competitively to tubulin. At the same low concentrations, vinblastine almost completely inhibited Tau- and MAP2-mediated tubulin assembly. At higher concentrations of 10 to 40 microM, a more striking difference was observed between the actions of the two drugs. Maytansine very effectively inhibited tubulin assembly promoted by either Tau or MAP2. Vinblastine also had this effect on MAP2-mediated tubulin assembly but in the presence of Tau induced extensive tubulin aggregation into spirals. In addition maytansine strongly inhibited vinblastine-induced Tau-dependent tubulin aggregation into spiral polymers. Even at very low concentrations, maytansine completely inhibited the effect of very high concentrations of vinblastine. These results very strongly suggest that the binding sites of maytansine and vinblastine on the tubulin molecule overlap and that the changes that they probably induce in the conformation of this molecule are markedly different, at least in the presence of microtubule-associated proteins.

Novel actions of the antitumor drugs vinflunine and vinorelbine on microtubules.

Vinflunine is a novel Vinca alkaloid presently in Phase I clinical trials. In preclinical studies, it exhibited superior antitumor activity to that of other Vinca alkaloids, including vinorelbine from which it was synthetically derived. Vinca alkaloids appear to inhibit cell proliferation by affecting the dynamics of spindle microtubules. Here we have analyzed the effects of vinflunine and vinorelbine on microtubule dynamic instability and treadmilling and found that these newer drugs exert effects on microtubule dynamics that differ significantly from those of the classic Vinca alkaloid, vinblastine. The major effects of vinflunine and vinorelbine on dynamic instability were a slowing of the microtubule growth rate, an increase in growth duration, and a reduction in shortening duration. In marked contrast to the action of vinblastine, they neither reduced the rate of shortening nor increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably. In addition, vinflunine and vinorelbine suppressed treadmilling, but less strongly than vinblastine. The diverse actions of these drugs on microtubules are likely to produce different effects on mitotic spindle function, leading to different effects on cell cycle progression and cell killing. Nontumor cells with normal checkpoint proteins may tolerate the relatively less powerful inhibitory effects of vinflunine and vinorelbine on microtubule dynamics better than the more powerful effects of vinblastine. Thus the unique constellation of effects of vinflunine and vinorelbine on dynamic instability and treadmilling may contribute to their superior antitumor efficacies.

Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'.

Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis. We show for the first time that paclitaxel acts directly or mitochondria isolated from human cancer cells. In isolated yeast mito chondria, paclitaxel (15 microM) induced an 18% increase in the respiration rate, with no concomitant release of cytochrome c. In isolated neuroblas toma mitochondria, paclitaxel (10-100 microM) induced a 27-72% release o cytochrome c. Release was prevented by cyclosporin A, suggesting the involvement of the permeability transition pore. Doxorubicin did no induce cytochrome c release, whereas vinorelbine, another antimicrotu bule agent, did. Thus, antimicrotubule agents can directly affect mito chondria to induce apoptosis.

Identification of a distinct class of vinblastine binding sites on microtubules.

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.

Microtubule dynamics: taking aim at a moving target.

Antitumor drugs of the vinca alkaloid and taxane classes function by suppressing the dynamics of microtubules in spindles, blocking cell division at metaphase. The drugs bind to various sites on the tubulin dimer and at different positions within the microtubule, suggesting that there are many unexplored targets for the design of novel drugs of this type.

Effects of gas tension on epidermal keratinocyte DNA synthesis and prostaglandin production.

The gas phase partial pressure of O2 (PO2) overlying mouse keratinocyte cultures controls the rate of DNA synthesis of these cells by an undefined mechanism. In these studies, both PO2 and PCO2 tensions overlying primary cell cultures were varied within the physiologic range. The prostaglandin (PG) production of cells grown under several varying gas tensions was then determined using radioimmune assay. The cultures were grown under a PO2 of either 7.5% (physiologic for in vivo epidermis) or 21% (atmospheric; culture conditions and wound healing) for 5 days. The PCO2 was either 5 or 10%, 2 CO2 tensions routinely used in tissue culture studies. DNA synthesis was quantitated using [3H]dThd uptake into DNA and autoradiography. The PGE2 and PGF2 alpha syntheses by the cultures over specific time periods were determined. Changing the PO2 from 21 to 7.5% decreased the rate of DNA synthesis, while PG production remained constant. When the PCO2 was varied from 5 to 10%, keratinocyte DNA synthesis remained unchanged but PG production was markedly stimulated. The PCO2 effect on PG production was greatest at the highest oxygen tension. The data indicated that under this set of variables, PG production by keratinocytes is not directly related to the rate of DNA synthesis of the cells. Apparently the amount of oxygen in the gas phase can have a permissive effect on epidermal keratinocyte PG production.