Mechanisms Involving DNA Damage Research Articles

Translational Insights into Molecular Mechanisms of Chemical Hepatocarcinogenesis for Improved Human Risk Assessment

Background: Hepatocellular carcinoma (HCC) is a prevalent liver cancer with major risk factors being hepatitis viral infections, alcohol, non-alcoholic fatty liver disease, and aflatoxin exposure. Both genotoxic and non-genotoxic agents can induce HCC through mechanisms involving DNA damage, oxidative stress, chronic inflammation, and disrupted signaling pathways like MAPK/ERK, PI3K/AKT, WNT/β-catenin and PPARα. While rodent assays are utilized to detect potential chemical hepatocarcinogens, species differences in pathways like PPARα and CAR/PXR activation impact human risk assessment. Purpose: This analysis provides an updated, critical examination of species concordance in mechanisms of hepatic carcinogenesis to inform human safety assessment of rodent liver tumor findings. Main Body: Rodent assays including 2-year bioassays, transgenic models, and short-term studies detect liver tumors through lifetime exposure or early biomarkers. However, rodent-specific PPARα and CAR/PXR activation, along with human risk factors like hepatitis, highlight key interspecies differences. Determining mode of action relevance requires evaluating mechanistic validity and pivotal key events leading to tumors across species. Non-genotoxic compounds eliciting rodent liver tumors can activate PPARα, CAR/PXR, and other pathways triggering increased cell replication; but downstream signaling may differ in human liver. Understanding applicability of these mechanisms in humans as well as incorporating human risk factors into experimental models is critical for accurate risk assessment. Conclusion: In summary, elucidating conserved versus divergent molecular mechanisms of hepatic carcinogenesis between rodents and humans is essential for appropriately interpreting rodent findings and safeguarding human health through science-based risk assessment frameworks and regulatory decision-making processes around potential chemical hazards.

Abstract A070: Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition

Abstract Inhibitors of PARP have been shown to sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation therapy through mechanisms involving DNA damage, replication stress, and PARP trapping independent of BRCA1/2 mutation status. These mechanisms have the potential to activate innate immune signaling leading to Type I Interferon (T1IFN) production. Thus, we hypothesized that the clinical PARP1/2 inhibitor olaparib would potentiate radiation-induced T1IFN to activate an anti-tumoral immune response and sensitize resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and signaling, tumor growth, sensitivity to PD-L1 immunotherapy, and the tumor immune microenvironment in mouse models of PDAC. We found that clinically achievable concentrations of olaparib significantly enhanced radiation-induced T1IFN production measured by both an IFNβ promoter-driven reporter and Ifnβ mRNA levels. Additionally, we found that the downstream effects of radiation-induced T1IFN signaling, including PD-L1 and MHC-I cell surface expression and induction of interferon stimulated genes including Cxcl9 and Cxcl10, were enhanced by olaparib. Co-treatment with a T1IFN receptor blocking antibody diminished these effects supporting the requirement for T1IFN-dependent signaling for the innate immune effects of radiation and olaparib. In vivo treatment with olaparib and radiation sensitized tumors to anti-PD-L1 therapy with a 40% complete response rate. These therapeutic responses led to an adaptive long term immunogenic memory evidenced by reduced engraftment following tumor rechallenge. Single-cell RNA sequencing revealed that treatment with olaparib, radiation and anti-PD-L1 augmented an anti-tumoral CD8+ T cell immune response, which is supported by an increase in CD8+ effector T cells and a decrease in CD8+ dysfunctional T cells. The necessity of functional CD8+ T cells to therapy efficacy in vivo was further reflected by a significantly reduced therapeutic combinational efficacy using a CD8+ T cell blocking antibody. Taken together, the results of this study show that the combination of olaparib, radiation and anti-PD-L1 induce an anti-tumoral immune response mediated by both T1IFN and CD8+ T cells. The results of this study provide the rationale for a phase 1 clinical trial (NCT05411094) combining dose escalated olaparib with durvalumab and radiotherapy in patients with locally advanced pancreatic cancer. Citation Format: Victoria M Valvo, Qiang Zhang, Long Jiang, Ashkay Tate, Michael D Green, Meredith A Morgan. Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A070.

Resveratrol Derivative Exhibits Marked Antiproliferative Actions, Affecting Stemness in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the deadliest malignancies, with an increasing incidence and limited response to current therapeutic options. Therefore, more effective and low-toxic agents are needed to improve PC patients' outcomes. Resveratrol (RSV) is a natural polyphenol with multiple biological properties, including anticancer effects. In this study, we explored the antiproliferative activities of newly synthetized RSV analogues in a panel of PC cell lines and evaluated the physicochemical properties of the most active compound. This derivative exhibited marked antiproliferative effects in PC cells through mechanisms involving DNA damage, apoptosis induction, and interference in cell cycle progression, as assessed using flow cytometry and immunoblot analysis of cell cycle proteins, PARP cleavage, and H2AX phosphorylation. Notably, the compound induced a consistent reduction in the PC cell subpopulation with a CD133+EpCAM+ stem-like phenotype, paralleled by dramatic effects on cell clonogenicity. Moreover, the RSV derivative had negligible toxicity against normal HFF-1 cells and, thus, good selectivity index values toward PC cell lines. Remarkably, its higher lipophilicity and stability in human plasma, as compared to RSV, might ensure a better permeation along the gastrointestinal tract. Our results provide insights into the mechanisms of action contributing to the antiproliferative activity of a synthetic RSV analogue, supporting its potential value in the search for effective and safe agents in PC treatment.

Absence of a robust mitotic timer mechanism in early preimplantation mouse embryos leads to chromosome instability

Preimplantation embryos often consist of a combination of euploid and aneuploid cells, suggesting that safeguards preventing the generation and propagation of aneuploid cells in somatic cells might be deficient in embryos. In somatic cells, a mitotic timer mechanism has been described, in which even a small increase in the duration of M phase can cause a cell cycle arrest in the subsequent interphase, preventing further propagation of cells that have undergone a potentially hazardously long M phase. Here, we report that cell divisions in the mouse embryo and embryonic development continue even after a mitotic prolongation of several hours. However, similar M-phase extensions caused cohesion fatigue, resulting in prematurely separated sister chromatids and the production of micronuclei. Only extreme prolongation of M phase caused a subsequent interphase arrest, through a mechanism involving DNA damage. Our data suggest that the simultaneous absence of a robust mitotic timer and susceptibility of the embryo to cohesion fatigue could contribute to chromosome instability in mammalian embryos. This article has an associated 'The people behind the papers' interview.

Novel Pt(IV) Prodrugs Displaying Antimitochondrial Effects.

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.

Pt(IV) pro-drugs with an axial HDAC inhibitor demonstrate multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance in A2780/A2780cis cells

Epigenetic agents such as histone deacetylase (HDAC) inhibitors are widely investigated for use in combined anticancer therapy and the co-administration of Pt drugs with HDAC inhibitors has shown promise for the treatment of resistant cancers. Coordination of an HDAC inhibitor to an axial position of a Pt(IV) derivative of cisplatin allows the combination of the epigenetic drug and the Pt chemotherapeutic into a single molecule. In this work we carry out mechanistic studies on the known Pt(IV) complex cis,cis,trans-[Pt(NH3)2Cl2(PBA)2] (B) with the HDAC inhibitor 4-phenylbutyrate (PBA) and its derivatives cis,cis,trans-[Pt(NH3)2Cl2(PBA)(OH)] (A), cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Bz)] (C), and cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Suc)] (D) (Bz = benzoate, Suc = succinate). The comparison of the cytotoxicity, effect on HDAC activity, reactive oxygen species (ROS) generation, γ-H2AX (histone 2A-family member X) foci generation and induction of apoptosis in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells shows that A – C exhibit multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance.

An Anticancer PtIV Prodrug That Acts by Mechanisms Involving DNA Damage and Different Epigenetic Effects.

Dual- or multi-action PtIV prodrugs represent a new generation of platinum anticancer drugs. The important property of these PtIV prodrugs is that their antitumor action combines several different mechanisms owing to the presence of biologically active axial ligands. This work describes the synthesis and some biological properties of a "triple-action" prodrug that releases in cancer cells cisplatin and two different epigenetically acting moieties, octanoate and phenylbutyrate. It is demonstrated, with the aid of modern methods of molecular and cellular biology and pharmacology, that the presence of three different functionalities in a single molecule of the PtIV prodrug results in a selective and high potency in tumor cells including those resistant to cisplatin [the IC50 values in the screened malignant cell lines ranged from as low as 9 nm (HCT-116) to 74 nm (MDA-MB-231)]. It is also demonstrated that cellular activation of the PtIV prodrug results in covalent modification of DNA through the release of the platinum moiety accompanied by inhibition of the activity of histone deacetylases caused by phenylbutyrate and by global hypermethylation of DNA by octanoate. Thus, the PtIV prodrug introduced in this study acts as a true "multi-action" prodrug, which is over two orders of magnitude more active than clinically used cisplatin, in both 2D monolayer culture and 3D spheroid cancer cells.

Abstract 4841: Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex

Abstract Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target. Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.

MP68-06 FORMATION OF DOUBLE-STRANDED RNA ACTIVATES STRESS KINASES AND INITIATES CYTOSKELETON ORGANIZATION AND CELL DEGENERATION IN BLADDER ISCHEMIA

INTRODUCTION AND OBJECTIVES: Double-stranded RNA (ds-RNA) is RNA with two complementary strands, which is not readily detected in normal tissues but is produced as a replicative intermediate in cellular stress conditions. By binding to and activating stress kinases and inducing mRNA degradation, ds-RNA can serve as a potent stimulus to cellular and subcellular structural modifications. We examined the formation and structural consequences of ds-RNA in chronic bladder ischemia. METHODS: A rat bladder ischemia model was developed by creating aorto-iliac arteries atherosclerosis. After 8 weeks, the animals underwent metabolic cage studies then hemodynamic measurements and cystometrograms (CMG) were obtained. Bladder tissues were processed for RT-PCR of ds-RNA precursor Alu-RNA, dot blot analysis of ds-RNA formation, western blotting of ds-RNA-dependent protein kinase (PKR), mass spectrometry, ELISA of stress markers, and transmission electron microscopy (TEM). RESULTS: Chronic bladder ischemia increased micturition frequency, decreased voided volume and led to detrusor instability. Ischemia upregulated Alu-RNA expression and led to the formation of ds-RNA and activation of the stress kinase PKR in the bladder. These changes were associated with cellular lipid and protein stress markers. Accumulation of ds-RNA and phosphorylation of PKR resulted in DNA damage, loss of proteins, and activation of degenerative pathways. Mass spectrometry and Gen Ontology analysis suggested cytoskeleton organization and degenerative activities mediated by ubiquitination and increased proteolysis, peptidase and hydrolase activities. EM confirmed cytoskeleton organization and cell degeneration characterized by swollen mitochondria with disrupted membrane and decreased granules, swollen elongated ER, collagen invasion of smooth muscle cells and nerve fibers, sporadic loss of epithelial mucosal membrane, twisted smooth muscle cells, diffused vacuolization, and loss of neural structural integrity. CONCLUSIONS: We report the formation of ds-RNA and subsequent phosphorylation of PKR in bladder ischemia. Activation of dsRNA/PKR pathway in the bladder resulted in widespread structural modifications via mechanisms involving DNA damage, mRNA degradation and loss of proteins. Our data suggest a close link between dsRNA/PKR pathway, proteolysis, cytoskeleton organization and loss of cellular structural integrity. Formation of ds-RNA and activation of PKR may precede cellular ultrastructural deterioration and play a central role in loss of smooth muscle cells and neurodegeneration in the ischemic overactive bladder.

Cordyceps militaris (L.) Link Fruiting Body Reduces the Growth of a Non-Small Cell Lung Cancer Cell Line by Increasing Cellular Levels of p53 and p21.

Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies.

Non-thermal atmospheric pressure plasma induces apoptosis in oral cavity squamous cell carcinoma: Involvement of DNA-damage-triggering sub-G1 arrest via the ATM/p53 pathway

Recent advances in physics have made possible the use of non-thermal atmospheric pressure plasma (NTP) in cancer research. Although increasing evidence suggests that NTP induces death of various cancer cell types, thus offering a promising alternative treatment, the mechanism of its therapeutic effect is little understood. In this study, we report for the first time that NTP led to apoptotic cell death in oral cavity squamous cell carcinoma (OSCC). Interestingly, NTP induced a sub-G1 arrest in p53 wild-type OSCCs, but not in p53-mutated OSCCs. In addition, NTP increased the expression levels of ATM, p53 (Ser 15, 20 and 46), p21, and cyclin D1. A comet assay, Western blotting and immunocytochemistry of γH2AX suggested that NTP-induced apoptosis and sub-G1 arrest were associated with DNA damage and the ATM/p53 signaling pathway in SCC25 cells. Moreover, ATM knockdown using siRNA attenuated the effect of NTP on cell death, sub-G1 arrest and related signals. Taken together, these results indicate that NTP induced apoptotic cell death in p53 wild-type OSCCs through a novel mechanism involving DNA damage and triggering of sub-G1 arrest via the ATM/p53 pathway. These findings show the therapeutic potential of NTP in OSCC.

Novel Nitrobenzazolo[3,2-a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Changes, and Mitochondrial Permeabilization.

This study reports the capacity of three nitro substituted benzazolo[3,2-a]quinolinium salts NBQs: NBQ 95 (NSC-763304), NBQ 38 (NSC 763305), and NBQ 97 (NSC-763306) as potential antitumor agents. NBQ’s are unnatural alkaloids possessing a positive charge that could facilitate interaction with cell organelles. The anticancer activities of these compounds were evaluated through the National Cancer Institute (NCI) 60 cell line screening which represents diverse histologies. The screening was performed at 10 µM on all cell lines. Results from the NCI screening indicated cytotoxicity activity on six cell lines. In order to explore a possible mechanism of action, a detailed biological activity study of NBQ 95 and NBQ 38 was performed on A431 human epidermoid carcinoma cells to determine an apoptotic pathway involving, cell cycle changes, DNA fragmentation, mutations, mitochondrial membrane permeabilization and caspases activation. DNA fragmentation, cell cycle effects, mutagenesis, mitochondrial permeabilization and activation of caspases were determined by fluorimetry and differential imaging. Our data showed that A431 growth was inhibited with an average IC50 of 30 µM. In terms of the mechanism, these compounds interacted with DNA causing fragmentation and cell cycle arrest at sub G0/G1 stage. Mutagenesis was higher for NBQ 38 and moderate for NBQ 95 Mitochon-drial permeabilization was observed with NBQ 38 and slightly for NBQ 95. Both compounds caused activation of Caspases 3 and 7 suggesting an apoptotic cell death pathway through an intrinsic mechanism. This study reports evidence of the toxicity of these novel compounds with overlapping structural and mechanistic similarities to ellipticine, a known anti-tumor compound.

Injury-induced spinal motor neuron apoptosis is preceded by DNA single-strand breaks and is p53- and Bax-dependent.

The mechanisms of injury-induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve-spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7-14 days postlesion. This motor neuron apoptosis is blocked in bax(-/-) and p53(-/-) mice. Single-cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single-strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4-5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase-3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase-3. We conclude that adult motor neuron death after nerve avulsion is DNA damage-induced, p53- and Bax-dependent apoptosis.

In vitro neurotoxic and DNA-damaging properties of nitrogen mustard.

Sulfur mustard and nitrogen mustard (HN2) are reported to produce neurobehavioral and neuropathological changes in animals and humans, but the mechanisms are unknown. We examined the cytotoxic properties of HN2 in cultures of dividing and post-mitotic neurons and astrocytes, which comprise the majority of cells in the central nervous system. Cultures of rat cerebellar astrocytes, post-mitotic granule cell neurons or dividing and terminally differentiated human SY5Y neuroblastoma cell cultures were treated with various concentrations of HN2 for 24 h. After treatment, culture medium was removed, the cell monolayer was incubated for 30 min with calcein-AM (green, live cells) and propidium iodide (red, dead cells) in control medium, the fluorochrome-containing medium was removed and replaced with control medium and cell density and viability were examined by fluorescence and light microscopy. Extensive cell loss (>90%) was observed in rat neuronal and SY5Y neuroblastoma cell cultures treated with 10 microM HN2, whereas cell loss was similar to controls in comparably treated astrocyte cultures. The DNA from HN2-treated cultures of rat neurons and SY5Y neuroblastoma cells was examined by high-performance liquid chromatography with electrochemical detection for the major HN2 DNA adduct N-(2-hydroxyethyl)-N[2-(7-guaninyl)ethyl]methylamine (GMOH). GMOH was detected in rat neuronal (85 fmol microg(-1) DNA) and SY5Y neuroblastoma cell cultures (46 fmol microg(-1) DNA) treated with 10 microM HN2 for 24 h, but was not detected in comparably treated astrocyte cell cultures. These findings are consistent with HN2 preferentially targeting neurons in vivo, possibly through a mechanism involving DNA damage.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists

Objective To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. Design In vitro explant culture of leiomyoma cells. Setting University research group. Sample Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. Methods The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase–olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Main outcome measures Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Results Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 β-oestradiol. Conclusion Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists.

To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. In vitro explant culture of leiomyoma cells. University research group. Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase-olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 beta-oestradiol. Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.