DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2). Somewhat higher concentrations resulted in substantial S-phase arrest, and at increasing concentrations, complete cell cycle arrest in G(1) was observed without the appearance of a sub-G(1) cell population. Neither inhibition of cell growth nor cell cycle arrest appeared to be dependent on ataxia and Rad-related kinase expression. DNA damage checkpoint proteins including p53, chk1, and chk2 were differentially activated by hedamycin depending on the concentration and duration of treatment. The level of downstream cell cycle regulators such as cdc25A, E2F1, cyclin E, and p21 were also altered under conditions that induced cell cycle arrest, but atypically, p21 overexpression was observed only in S-phase-arrested cells. Apoptotic indicators were only observed at moderate hedamycin concentrations associated with S-phase arrest, while increasing concentrations, when cells were arrested in G(1), resulted in a reduction of these signals. Taken together, the responses of cells to hedamycin are distinct with regard to its effect on cell cycle but also in the unusual concentration-dependent manner of activation of DNA damage and cell cycle checkpoint proteins as well as the induction of apoptotic-associated events.     

Ultraviolet Radiation in Skin Ageing and Carcinogenesis: The Role of Retinoids for Treatment and Prevention

The mechanisms of UV-induced ageing and carcinogenesis of the skin have been elucidated in animals and humans, and both UVB and UVA radiation have been shown to have deleterious effects on the skin. Thus the use of solaria which deliver mostly UVA radiation is not safe. There is also an increased risk of ageing when using therapeutic UV sources. UV radiation is beneficial in many cases of skin disorders such as psoriasis, atopic eczema, acne and pruritus. Nevertheless by careful patient selection and follow-up the risks of UV can be minimised when treating patients with artificial UV radiation. During recent years there has been intensive research into the development of agents which prevent harmful effects of radiation. The retinoids are particularly interesting as they enhance skin repair after UV damage, have an anticarcinogenic effect and are effective for treating precancerous lesions such as solar keratosis and as adjuvant therapy for skin cancers. Topical retinoids are already used for the treatment of actinic skin damage, and systemic retinoids are also used in certain groups of patients who have an increased risk of contracting skin cancers such as xeroderma pigmentosum.     

Ultraviolet radiation in skin ageing and carcinogenesis: the role of retinoids for treatment and prevention.

The mechanisms of UV-induced ageing and carcinogenesis of the skin have been elucidated in animals and humans, and both UVB and UVA radiation have been shown to have deleterious effects on the skin. Thus the use of solaria which deliver mostly UVA radiation is not safe. There is also an increased risk of ageing when using therapeutic UV sources. UV radiation is beneficial in many cases of skin disorders such as psoriasis, atopic eczema, acne and pruritus. Nevertheless by careful patient selection and follow-up the risks of UV can be minimised when treating patients with artificial UV radiation. During recent years there has been intensive research into the development of agents which prevent harmful effects of radiation. The retinoids are particularly interesting as they enhance skin repair after UV damage, have an anticarcinogenic effect and are effective for treating precancerous lesions such as solar keratosis and as adjuvant therapy for skin cancers. Topical retinoids are already used for the treatment of actinic skin damage, and systemic retinoids are also used in certain groups of patients who have an increased risk of contracting skin cancers such as xeroderma pigmentosum.     

Acetyl-11-keto-β-boswellic acid triggers premature senescence via induction of DNA damage accompanied by impairment of DNA repair genes in hepatocellular carcinoma cells in vitro and in vivo

Cellular senescence, a state of irreversible growth arrest, occurs in all somatic cells and causes the cells to exhaust replicative capacity. Recently, cellular senescence has been emerging as one of the principal mechanisms of tumor suppression, which can be induced by low doses of therapeutic drugs in cancer cells. Acetyl-11-keto-β-boswellic acid (AKBA), an active ingredient isolated from the plant Boswellia serrata, has been identified to induce apoptosis in hepatocellular carcinoma (HCC) cells. In this study, we found that low concentrations of AKBA treatment triggered cell growth arrest at G0/G1 phase with features of premature cellular senescence phenotype in both HCC cell lines HepG2 and SMMC7721, as observed by enlarged and flattened morphology, significant increase in cells with senescence-associated β-galactosidase staining, and decrease in cell proliferation and DNA synthesis. Furthermore, cellular senescence induced by AKBA occurred via activation of DNA damage response and impairment of DNA repair, as evidenced by strong induction of γH2AX and p53, and downregulated expressions of multiple DNA repair associated genes. Induction of p53 by AKBA caused a significant increase in p21^CIP1, which had a critical involvement in the induction of cellular senescence. Additionally, in vivo study demonstrated that induction of senescence contributed to the anticancer efficacy of AKBA. Therefore, our findings suggested that induction of premature senescence by AKBA through DNA damage response accompanied by impairment of DNA repair genes defines a novel mechanism contributing to its growth suppression in HCC cells.     

The DNA minor groove-alkylating cyclopropylpyrroloindole drugs adozelesin and bizelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells

As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses. Low adozelesin concentrations (e.g., 0.5 nM) induced a transient S-phase block and cell cycle arrest in G(2)-M, as well as increased induction of p53 and p21, whereas a high drug concentration (e.g., 2.5 nM) caused apoptosis but no p21 induction. In contrast, both low and high bizelesin concentrations enhanced p53 and p21 induction and triggered G(2)-M cell cycle arrest and eventual senescence without significant apoptotic cell death. However, in cells lacking p21, bizelesin, as well as adozelesin, triggered apoptosis, indicating that p21 was crucial to sustained bizelesin-induced G(2)-M arrest. Thus, despite similar abilities to alkylate DNA, the chemotherapeutic agents adozelesin and bizelesin caused a decrease in HCT116 tumor cell proliferation by different pathways (i.e., adozelesin induced apoptosis, and bizelesin induced senescence).     

Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.     

Electro-gene transfer to skin using a noninvasive multielectrode array

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection of plasmid DNA alone over 15 days. Furthermore, gene expression could be enhanced by increasing the size of the treatment area. Transgene-expressing cells were observed exclusively in the epidermal layer of the skin. In contrast to caliper or plate electrodes, skin EP with the MEA greatly reduced muscle twitching and resulted in minimal and completely recoverable skin damage. These results suggest that EP with MEA can be an efficient and non-invasive skin delivery method with less adverse side effects than other EP delivery systems and promising clinical applications.     

The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity

The chemotherapeutic agent temozolomide produces O(6)-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G(2) arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci. MRN foci formed significantly earlier than gamma-H2AX and 53BP1 foci that assembled in response to temozolomide-induced DNA DSBs. MRN foci formation was suppressed in cells that incurred lower levels of temozolomide-induced O6MG lesions and/or had decreased mismatch repair capabilities, suggesting that the MRN foci formed not in response to temozolomide-induced DSB but rather in response to mismatch repair processing of mispaired temozolomide-induced O6MG lesions. Consistent with this idea, the MRN foci colocalized with those of proliferating cell nuclear antigen (a component of the mismatch repair complex), and the MRN complex component Nbs1 coimmunoprecipitated with the mismatch repair protein Mlh1 specifically in response to temozolomide treatment. Furthermore, small inhibitory RNA-mediated suppression of Mre11 levels decreased temozolomide-induced G(2) arrest and cytotoxicity in a manner comparable to that achieved by suppression of mismatch repair. These data show that temozolomide-induced O6MG lesions, acted upon by the mismatch repair system, drive formation of the MRN complex foci and the interaction of this complex with the mismatch repair machinery. The MRN complex in turn contributes to the control of temozolomide-induced G(2) arrest and cytotoxicity, and as such is an additional determining factor in glioma sensitivity to DNA methylating chemotherapeutic drugs such as temozolomide.     

Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (gamma-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941-54].     

Topical retinoids in inflammatory acne: a retrospective, investigator-blinded, vehicle-controlled, photographic assessment

BACKGROUND: Despite published data showing the efficacy of topical retinoids in treating inflammatory acne, in clinical practice topical retinoids tend to be used most commonly for noninflammatory acne. OBJECTIVE: The goal of this study was to assess the efficacy of topical retinoids as monotherapy in inflammatory acne. METHODS: This retrospective, investigator-blinded,vehicle-controlled, photographic assessment study was conducted by 5 investigators. The investigators rated pretreatment and posttreatment photographs of patients who had participated in 12- or 15-week, double-blind comparisons of tazarotene 0.1% gel, adapalene 0.1% gel, tretinoin 0.1% microsponge, tretinoin 0.025% gel, and tazarotene 0.1% cream (vehicle). Acne severity was rated on a 7-point scale. A posttreatment increase or decrease of 1 grade was considered clinically meaningful; > or =2 grades was considered an even clearer measure of clinically significant improvement. Investigators also rated global response to treatment on a 7-point scale. A posttreatment increase of > or =2 grades was considered a clinically relevant improvement. RESULTS: Each of the 5 investigators rated photographs of 577 patients ( approximately 52% women, approximately 48% men; mean age, 18-20 years), for a total of 2885 evaluations (in addition to daily evaluations of the 20 control patients). The treatment groups consisted of tazarotene (252 patients, 1260 evaluations), adapalene (178 patients, 890 evaluations), tretinoin microsponge (47 patients, 235 evaluations), tretinoin gel (39 patients, 195 evaluations), and vehicle (61 patients, 305 evaluations). Inflammatory acne was improved with all 4 retinoids compared with vehicle. In 1905 evaluations in which pretreatment acne severity was grade > or =3 (mild to moderate), the incidences of clinically significant improvements in the tazarotene, adapalene, and tretinoin microsponge groups were 24%, 17%, and 21%, respectively (all, P < or = 0.001 vs vehicle [7%]). The difference in prevalence of clinically significant improvement was statistically similar between the tretinoin gel and vehicle groups. The incidences of clinically relevant improvement in global response to tazarotene, adapalene, tretinoin microsponge, and tretinoin gel were 36%, 34%, 31%, and 28%, respectively (P < or = 0.001, < or =0.001, < or =0.001, and < or =0.01, respectively, vs vehicle [17%]). CONCLUSIONS:: The results of this study suggest that topical retinoid monotherapy can achieve clinically significant improvements in inflammatory acne.     

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser(139) (gamma-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of gamma-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser(1981) was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation.     

Topical therapy for acne

Acne is a common problem in adolescents and young adults. The disorder is caused by abnormal desquamation of follicular epithelium that results in obstruction of the pilosebaceous canal. This obstruction leads to the formation of comedones, which can become inflamed because of overgrowth of Propionibacterium acnes. Topical retinoids such as tretinoin or adapalene are effective in many patients with comedonal acne. Patients with inflammatory lesions benefit from treatment with benzoyl peroxide, azelaic acid or topical antibiotics. Frequently, the use of comedonal and antibacterial agents is required.     

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications.     

Targeting tyrosine phosphorylation of PCNA inhibits prostate cancer growth

The proliferation cell nuclear antigen (PCNA) is a critical protein required for DNA replication in proliferating cells including cancer cells. However, direct inhibition of PCNA in cancer cells has been difficult due to the lack of targetable sites. We previously reported that phosphorylation of tyrosine 211 (Y211) on PCNA is important for the proliferative function of PCNA when this protein is associated with the chromatin in cancer cells. Here, we show that the Y211 phosphorylation of PCNA is a frequent event in advanced prostate cancer. To explore the potential of this signaling event in inhibition of cancer cell growth, we used a synthetic peptide, the Y211F peptide, which when present inhibits phosphorylation of Y211 on endogenous PCNA. Treatment with this peptide, but not a scrambled control peptide, resulted in S-phase arrest, inhibition of DNA synthesis, and enhanced cell death in a panel of human prostate cancer cell lines. In addition, treatment with the Y211F peptide led to decreased tumor growth in PC3-derived xenograft tumors in vivo in nude mice. Our study shows for the first time that PCNA phosphorylation at Y211 is a frequent and biologically important signaling event in prostate cancer. This study also shows a proof of concept that Y211 phosphorylation of PCNA may be used as a therapeutic target in prostate cancer cells, including cells of advanced cancers that are refractory to standard hormonal therapies.     

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss–induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a “pro-senescence” approach for cancer prevention and therapy.     

Flow cytometric DNA analysis of human endometrial carcinoma

Analysis of endometrial carcinoma of 15 cases and the normal endometrium from 12 control cases was made using flow cytometry. The proliferative activity and the ploidy level of the tumors were determined and a comparative study of the tumors classified by the nuclear grade of the tumor was made. In comparison with normal endometrium, endometrial carcinoma tissue had a significantly larger S-phase fraction of the cell cycle. Moreover, among the endometrial carcinoma cases, the percentage of S-phase cells was found to increase with increases in abnormalities of nuclear morphology. Endometrial carcinoma cases had higher levels of G2+M phase fractions than normal endometrium, but not significantly so. The proliferation index of endometrial carcinomas was significantly higher than that in normal endometrium, and it was found that proliferative activity became more robust the more irregular the nuclear morphology. With regard to the ploidy level, all of the normal endometria and Nuclear Grade 1 or 2 endometrial carcinoma cases showed a DNA distribution between diploid and tetraploid. In contrast, Nuclear Grade 3 cases could be subdivided into two groups, one which had a DNA distribution between diploid and tetraploid with an extremely high proliferation index and the other which had an aneuploid DNA stem line.     

Cytotoxicity of 5-Aza-2′-deoxycytidine against gastric cancer involves DNA damage in an ATM-P53 dependent signaling pathway and demethylation of P16

The DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) has increasingly attracted worldwide attention for its antineoplastic potential. The cytotoxitic mechanisms, however, especially, the relative contribution of silenced genes reactivation by demethylation and enzyme-DNA adduct formation to the efficacy of 5-Aza-CdR is still a crucial unresolved question. In this investigation, we demonstrated that 5-Aza-CdR treatment resulted in growth suppression in a concentration and time-dependent manner and G2 phrase arrest — hallmarks of a DNA damage response in gastric cancer AGS cells. Formation of DNA double-strand breaks, as monitored by comet assay was examined in an ATM (ataxia-telangiectasia mutated)-dependent manner based on the fact that PI3K inhibitor Wortmannin abolished the action of cytotoxicity of 5-Aza-CdR. Upon treatment with 5-Aza-CdR, ATM activation was clearly associated with P53 phosphorylation at Ser¹⁵, which was directly responsible for 5-Aza-CdR modified P21^Waf1/Cip1 expression. Further exploration revealed that demethylation of P16^INK4A correlated with the strikingly down-regulated expressions of DNA methyltransferase 3A as well as 3B was, at least in part, attributed to the cytotoxicity of 5-Aza-CdR in AGS cells. Conclusively, these results greatly enhance our understanding of the mechanisms of cytotoxicity of 5-Aza-CdR and strongly provide the preclinical rationale for an assessment of 5-Aza-CdR to ameliorate patient outcome with gastric cancer.     

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.