BLM Cells Research Articles

Extracellular vesicles from highly invasive melanoma subpopulations increase the invasive capacity of less invasive melanoma cells through mir-1246-mediated inhibition of CCNG2

Invasive growth is a critical process in tumor progression, requiring the activation of various molecular processes in tumor cells at the invasive front. Intercellular communication between heterogeneous tumor cells enhances cellular activation and adaptation to specific microenvironments. One mechanism of intercellular communication is the delivery of miRNAs through tumor cell-derived extracellular vesicles (EVs). In this context we have observed that conditioned media from a highly invasive cell subpopulation (BLM-HI) enhances the invasive capacity of the parental cell line (BLM). Therefore, we hypothesized that this complex change of cellular behavior is influenced by EV-transported miRNAs. The treatment of BLM cells with EVs derived from BLM-HI cells resulted in a significantly enhanced invasive capacity, as observed in Matrigel-embedded spheroids and in 2D Boyden chamber assays, with a dose-dependent effect. Conversely, the invasive capacity of BLM cells was reduced when secretion of EVs was inhibited by a sphingomyelinase inhibitor. To investigate the molecular mechanisms behind this effect, we performed next-generation sequencing and identified an enrichment of miR-1246 in these EVs. In functional analyses we demonstrated that both the EV mediated delivery of miR-1246 as well as overexpression contributes to the enhanced invasiveness of BLM cells. We identified a binding site of miR-1246 in the 3’UTR of cyclin G2 (CCNG2) and demonstrated direct binding by a luciferase reporter assay.Increased expression of CCNG2 has been associated with cancer metastasis and poor patient outcomes in other malignancies. Our study demonstrates that intercellular communication contributes to the transfer of properties, such as increased invasive capacity, between heterogeneous melanoma cells via EV-transported miRNAs.

Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures.

Melanoma is characterized by high metastatic potential favored by the epithelial-to-mesenchymal transition (EMT), leading melanoma cells to exhibit a spectrum of typical EMT markers. This study aimed to analyze the expression of EMT markers in A375 and BLM melanoma cell lines cultured in 2D monolayers and 3D spheroids using morphological and molecular methods. The expression of EMT markers was strongly affected by 3D arrangement and revealed a hybrid phenotype for the two cell lines. Indeed, although E-cadherin was almost undetectable in both A375 and BLM cells, cortical actin was detected in A375 2D monolayers and 3D spheroids and was strongly expressed in BLM 3D spheroids. The mesenchymal marker N-cadherin was significantly up-regulated in A375 3D spheroids while undetectable in BLM cells, but vimentin was similarly expressed in both cell lines at the gene and protein levels. This pattern suggests that A375 cells exhibit a more undifferentiated/mesenchymal phenotype, while BLM cells have more melanocytic/differentiated characteristics. Accordingly, the Zeb1 and 2, Slug, Snail and Twist gene expression analyses showed that they were differentially expressed in 2D monolayers compared to 3D spheroids, supporting this view. Furthermore, A375 cells are characterized by a greater invasive potential, strongly influenced by 3D arrangement, compared to the BLM cell line, as evaluated by SDS-zymography and TIMPs gene expression analysis. Finally, TGF-β1, a master controller of EMT, and lysyl oxidase (LOX), involved in melanoma progression, were strongly up-regulated by 3D arrangement in the metastatic BLM cells alone, likely playing a role in the metastatic phases of melanoma progression. Overall, these findings suggest that A375 and BLM cells possess a hybrid/intermediate phenotype in relation to the expression of EMT markers. The former is characterized by a more mesenchymal/undifferentiated phenotype, while the latter shows a more melanocytic/differentiated phenotype. Our results contribute to the characterization of the role of EMT in melanoma cells and confirm that a 3D cell culture model could provide deeper insight into our understanding of the biology of melanoma.

BLM Sumoylation Is Required for Replication Stability and Normal Fork Velocity During DNA Replication.

BLM is sumoylated in response to replication stress. We have studied the role of BLM sumoylation in physiologically normal and replication-stressed conditions by expressing in BLM-deficient cells a BLM with SUMO acceptor-site mutations, which we refer to as SUMO-mutant BLM cells. SUMO-mutant BLM cells exhibited multiple defects in both stressed and unstressed DNA replication conditions, including, in hydroxyurea-treated cells, reduced fork restart and increased fork collapse and, in untreated cells, slower fork velocity and increased fork instability as assayed by track-length asymmetry. We further showed by fluorescence recovery after photobleaching that SUMO-mutant BLM protein was less dynamic than normal BLM and comprised a higher immobile fraction at collapsed replication forks. BLM sumoylation has previously been linked to the recruitment of RAD51 to stressed forks in hydroxyurea-treated cells. An important unresolved question is whether the failure to efficiently recruit RAD51 is the explanation for replication stress in untreated SUMO-mutant BLM cells.

In-Vitro Effects of Active Vitamin-D3 and Vitamin-A on Human Melanoma (BLM and1205Lu) Cell Growth

Though UV ray in the Sun light is essential for vitamin-D3 formation, yet UV rays can lead to skin cancer. Lack of vitamin-D3 also can lead to cancer. Hence, we decided to study the effect of vitamin-D3 on human melanoma cell models. Our aims were to study the in-vitro effects of vitamin-D3 and vitamin-A on human melanoma (BLM, 1205Lu) cell growth, as vitamins D3 and A are important for a healthy skin. Initially dose-curve (100 nM to 100 μM concentration) study was carried out with vitamin-D3 and vitamin-A (retinoic acid) on BLM cells to determine the optimal concentrations of vitamins-D3 and A for co-incubation with progesterone (50 μM) and RU-486 (50 μM). Supernatants from the treated cells were subjected to Elisarray. Vitamin-D3 and vitamin-A showed a dose-dependent decrease in cell growth. Based on the dose-curve, it was decided to use 25 μM (at 57% cell growth) of vitamin-D3 and 50 μM (at 55% cell growth) of vitamin-A for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of BLM cell growth (20%). Similarly co-incubation of vitamin-D3 with progesterone (33% cell growth) and with RU-486 (28% cell growth) as well as co-incubation of vitamin-A with progesterone (31% cell growth) and RU-486 (18% cell growth) showed an additive effect on the decrease of BLM cell growth. Based on the BLM co-incubation studies, we decided to repeat the studies on 1205Lu cells. So, 25 μM (at 34% cell growth) of vitamin-D3 and 50 μM (at 44% cell growth) of vitamin-A were used for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of cell growth (22%). Though co-incubation of vitamin-D3 with progesterone did not show any difference in cell growth (51%), yet co-incubation with RU-486 showed a decrease in 1205Lu cell growth (21%). Similarly co-incubation of vitamin-A with progesterone (25% cell growth) and with RU-486 (23% cell growth) showed a synergistic effect on the decrease of 1205Lu cell growth. Conclusion: These studies suggested that combination of vitamins and steroids might be effective in decreasing melanoma cell growth. Hence, various combination of vitamins and steroids will be tested for their effect on melanoma cell growth in vitro in order to develop a combo drug treatment for melanoma.

Can Interleukin‐8 Alone be Responsible for Regulating Melanoma Growth & Metastasis? In‐vitro studies based on treatments of human melanoma cell lines (BLM and 1205Lu) with sex steroids, IL‐8, curcumin and active vitamin‐D3

Previous clinical studies indicated that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. But, the mechanism responsible for protection in menstruating females was not investigated. In addition, epidemiological data showed an increased male mortality in melanoma. Our in‐vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Elisarray showed that progesterone action was mediated by a specific suppression of pro‐inflammatory cytokine IL‐8. So, the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth was checked. AD and T also suppressed cell growth and IL‐8 secretion. But, addition of progesterone (10μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and IL‐8 suppression. This observation gave the idea of protective function of progesterone in melanoma, which was experimentally observed by a significant decrease in in‐vitro migration and adhesion functions. As steroids (P, AD, T) targeted IL‐8 for their actions, effect of vit‐D3 on melanoma cells was checked. Active form of vitamin‐D3 (25 μM) also targeted IL‐8 and decreased cell growth, suggesting IL‐8 could be regulating melanoma cell growth. In order to check whether IL‐8 regulated melanoma cell growth, IL‐8 was directly added to melanoma cells and endogenous IL‐8 was suppressed by 2 hrs pre‐treatment with curcumin. Addition of IL‐8 (1 ng/ml) to 1205Lu melanoma cells stimulated cell growth to 117%, whereas suppression of IL‐8 by curcumin (100 μM) pre‐treatment decreased cell growth to 24%. Similar results were obtained with BLM cells. This brought IL‐8 into focus as an important molecule regulating melanoma cell growth. Further in‐vitro experiments to restore curcumin (25 μM) pre‐treated melanoma cell growth (71.5%) by the addition of IL‐8 (100 ng/ml) resulted in a near complete restoration of cell growth (95.3%) in 1205Lu melanoma cells, suggesting IL‐8 could be the molecule regulating melanoma cell growth. Similar results were observed with BLM cells. In addition, in‐vivo conditional expression of IL‐8 in nude mouse by Dr. Singh et al., indicated involvement of IL‐8 in melanoma growth and metastasis.ConclusionBoth in‐vitro and in‐vivo studies indicated that IL‐8 alone could be responsible for regulating melanoma growth and metastasis. Hence, IL‐8 could be targeted to arrest melanoma growth and metastasis in‐vivo.

Increased elasticity of melanoma cells after low-LET proton beam due to actin cytoskeleton rearrangements

Cellular response to non-lethal radiation stress include perturbations in DNA repair, angiogenesis, migration, and adhesion, among others. Low-LET proton beam radiation has been shown to induce somewhat different biological response than photon radiation. For example, we have shown that non-lethal doses of proton beam radiation inhibited migration of cells and that this effect persisted long-term. Here, we have examined cellular elasticity and actin cytoskeleton organization in BLM cutaneous melanoma and Mel270 uveal melanoma cells. Proton beam radiation increased cellular elasticity to a greater extent than X-rays and both types of radiation induced changes in actin cytoskeleton organization. Vimentin level increased in BLM cells after both types of radiation. Our data show that cell elasticity increased substantially after low-LET proton beam and persisted long after radiation. This may have significant consequences for the migratory properties of melanoma cells, as well as for the cell susceptibility to therapy.

MON-312 Biochemical Basis of Steroid Hormones Action on Human Melanoma Cell Growth In-Vitro and Its Clinical Implication

: Clinical study showed that menstruating females were better protected in melanoma than post-menopausal women and men of any age. However, the study did not correlate with steroid status in females. Literature showed that progesterone (P) level varied in menstruating females between 1000 to 1500 ng/dL. Whereas in post-menopausal women, progesterone ranged between 20 to 100 ng/dL and in males between 27 - 90 ng/dL. Our earlier in-vitro research showed that progesterone significantly inhibited human melanoma (BLM) cell growth. So, it was hypothesized that progesterone could be the molecule protecting menstruating females in melanoma. Further research showed that androgens (androstenedione, AD and testosterone, T) also inhibited human melanoma cell growth in-vitro, but only at high concentrations (100 μM and 200 μM). Co-incubation of AD and T separately with P showed an additive effect on human melanoma cell growth inhibition with as low as 10 μM concentration of progesterone, suggesting the protective function of progesterone. As literature showed that progesterone action was mediated through cytokines, Elisarray was used to check the biochemical basis of P, AD and T actions on BLM cells. Elisarray showed that there was a specific suppression of IL-8 cytokine in the supernatants of AD, P and AD+P co-incubated cells compared to control cells. Decrease in IL-8 was quantitated by Elisa, which displayed a direct correlation with decrease in BLM cell growth. In order to confirm the above findings, all the experiments were repeated on another human melanoma 1205Lu cell line. There was a specific suppression of IL-8 in the supernatants, as shown by both Elisarray and IL-8 Elisa. To further consolidate the findings, action of another androgen T, P and T+P were checked on both BLM and 1205Lu cell lines. Again a specific suppression of IL-8 was observed in the supernatants by Elisarray. In addition, Kanda and Watanabe already showed that dihydrotestosterone and estrogen also suppressed IL-8 in melanoma cells. Conclusion: All the tested steroids specifically suppressed IL-8, resulting in the suppression of melanoma cell growth. So, IL-8 could be playing an important role in melanoma growth. In fact, our in-vitro study involving curcumin pre-treatment already showed that IL-8 was the molecule involved in the regulation of human melanoma cell growth. So, IL-8 could be considered as a target for melanoma treatment.

Biochemical Basis of Steroid Hormones Inhibition of Human Melanoma Cell Growth In‐Vitro & Its Implication on Melanoma Treatment

Skin, as an endocrine organ produced various hypothalamic, pituitary, adrenal and sex steroids. Steroids were not only important for healthy skin, but were also shown to influence locally and systemically. This raised the question whether melanoma was a hormone‐dependent cancer. But to the contrary, clinical study showed that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. Literature showed that progesterone (P) level varied in menstruating females between 1000 to 1500 ng/dL. Whereas in post‐menopausal women, progesterone ranged between 20 to 100 ng/dL and in males between 27 – 90 ng/dL. In addition, our in‐vitro research showed that progesterone significantly inhibited human melanoma (BLM) cell growth. So, it was hypothesized that progesterone could be protecting menstruating females in melanoma. Further research showed that androgens (androstenedione, AD and testosterone, T) also inhibited human melanoma cell growth in‐vitro, but only at high concentrations (100 μM and 200 μM). Co‐incubation of AD and T separately with P showed an additive effect on BLM cell growth inhibition, suggesting the protective function of P. In order to find out the biochemical basis of steroid hormones action, Elisarray was carried out on the supernatants. There was a specific suppression of IL‐8 cytokine in the supernatants of AD, P and AD+P co‐incubated cells compared to untreated control BLM cells. Same experiments were repeated on another human melanoma (1205Lu) cell line, which also showed a specific suppression of IL‐8 cytokine. The decrease in IL‐8 was quantitated by Elisa, which correlated with the decrease in melanoma cell growth. To further confirm, experiments with T, P and T+P were carried out on BLM and 1205Lu cell lines, which also showed a specific suppression of IL‐8 by Elisarray. In addition, Kanda and Watanabe already showed that dihydrotestosterone and estrogen also suppressed IL‐8 in melanoma cells.ConclusionSuppression of IL‐8 resulting in the suppression of melanoma cell growth, implied the important role of IL‐8 in regulating melanoma growth. But suppression of IL‐8 in turn by various steroids, indicated the local regulation by the steroids. Local regulation in turn indicated the role of steroids in regulating melanoma growth. Hence, balancing or restoring local steroid hormones along with targeted or immuno therapy could be an ideal treatment for melanoma.Support or Funding Information1) Start‐up grant to PR2) Biomedical Sciences research grant3) Warner/Fermaturo research grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leucurogin and melanoma therapy

Leucurogin is an ECD disintegrin-like protein, cloned from Bothrops leucurus venom gland. This new protein, encompassing the disintegrin region of a PIII metalloproteinase, is produced by recombinant technology and its biological and functional activity was partially characterized in this study. Biological activity was characterized in vitro using human fibroblasts. Functional activity of leucurogin was analysed in vitro and in vivo with murine B16F10 Nex-2 and human melanoma BLM cells. The results show that leucurogin inhibits cellular processes dependent on collagen type I. In a competition assay with collagen, leucurogin inhibits, in a dose-dependent manner, the adhesion of fibroblast to collagen. At 10 μM leucurogin reduces adhesion (40%) and migration (70%) of hFb and inhibits migration (32%) and proliferation (65%) of BLM cells. At 2.5 μM leucurogin inhibits 80% cell proliferation of B16F10 Nex-2 melanoma cells. At 4.8 μM leucurogin inhibits, in vitro, the vascular structures formation by endothelial cells by 66%. Leucurogin, injected intraperitoneally, i.p. (5 μg/animal, two-month old C57/Bl6 male mice) on alternate days for 15 days, inhibits lung metastasis of B16F10 Nex-2 cells by 70–75%. In the treatment of human melanoma, grafted intradermally in the nude mice flank, leucurogin (7.5 μg/kg in alternate days during 17 days) inhibits tumor growth by more than 40%. Leucurogin can be considered a promising agent for melanoma treatment.

Proton beam irradiation inhibits the migration of melanoma cells.

PurposeIn recent years experimental data have indicated that low-energy proton beam radiation might induce a difference in cellular migration in comparison to photons. We therefore set out to compare the effect of proton beam irradiation and X-rays on the survival and long-term migratory properties of two cell lines: uveal melanoma Mel270 and skin melanoma BLM.Materials and methodsCells treated with either proton beam or X-rays were analyzed for their survival using clonogenic assay and MTT test. Long-term migratory properties were assessed with time-lapse monitoring of individual cell movements, wound test and transpore migration, while the expression of the related proteins was measured with western blot.ResultsExposure to proton beam and X-rays led to similar survival but the quality of the cell colonies was markedly different. More paraclones with a low proliferative activity and fewer highly-proliferative holoclones were found after proton beam irradiation in comparison to X-rays. At 20 or 40 days post-irradiation, migratory capacity was decreased more by proton beam than by X-rays. The beta-1-integrin level was decreased in Mel270 cells after both types of radiation, while vimentin, a marker of EMT, was increased in BLM cells only.ConclusionsWe conclude that proton beam irradiation induced long-term inhibition of cellular motility, as well as changes in the level of beta-1 integrin and vimentin. If confirmed, the change in the quality, but not in the number of colonies after proton beam irradiation might favor tumor growth inhibition after fractionated proton therapy.

Cav‐1 Upregulation Slows Migration Velocity in BLM Melanoma

Caveolin‐1 (Cav‐1) is a membrane‐associated and cytosolic protein that influences a number of cellular signaling cascades. Changes in Cav‐1 expression levels are thought to be responsible for changes in metastatic behaviour of a number of cancers, including melanoma. However, the effect of changes in Cav‐1 expression level seem dependent on cell‐type and direction of expression change. In highly migratory, metastatic melanoma, Cav‐1 expression levels tend to be low. The purpose of this study was to determine whether Cav‐1 upregulation alters EMT markers, cell division or basal and CXCL‐8‐stimulated migration velocity in human BLM melanoma. Wild‐type BLM cells were stably transfected (Lipofectamine 2000, Invitrogen) with a Myc‐DDK‐tagged human Cav‐1 gene cloned into a pCMV6‐Entry vector (Origene, TrueOrf). Permanent selection was achieved with G418 (1 mg/ml) antibiotic. Cell extracts from wild‐type and transfected BLM cells were run on 10%‐PAGE gels and transferred to PVDF membranes. Appropriate 1° antibodies were used to confirm Cav‐1 upregulation and to test for the presence of the EMT marker N‐cadherin using infrared 2° antibody detection (LiCor). Proliferation of wild‐type and Cav‐1 transfected cells were measured using BrdU assay. Cells were plated in 96‐well plates and incubated for 24 hours in RPMI 1640, 10% FBS. BrdU was added to the cells and allowed to incubate for 12 hours. Cells were fixed, the DNA denatured, and mouse anti‐BrdU antibody added to detect the incorporated BrdU. BrdU uptake was measured using HRP‐linked 2° antibody and [TMB] detected at A450 nm using a spectrophotometer (Epoch Microplate Spectrophotometer). Migration velocity was measured in wild‐type and transfected cells using a wound‐healing assay. Cells were seeded at 250,000 cells per well in 24‐well plates and incubated overnight in serum free RPMI 1640 media. Wells were scratched with pipette tip and plated in RPMI 1640, 10% FBS. Wells received a range of unbound CXCL8 doses (0–100 ng/ml). Wound images were collected at 15 min intervals and quantified at 4 hour intervals using ImageJ. Immunoblot analysis confirmed increased expression of Myc‐DDK‐tagged Cav‐1 in the transfected BLM cells. Immunohistochemical staining also confirmed increased expression of Cav‐1 in the transfected cells and showed that subcellular distribution of Cav‐ 1 is similar in the wild‐type and transfected cells. The EMT marker N‐cadherin, which was largely absent in the wild‐type cells, showed increased expression in the Cav‐1 transfected BLMs. BrdU assay shows that there is no significant difference in cell division over 12 hours between wild‐type and Cav‐1 transfected cells by Student's t‐test (p = 0.54). Migration velocity by wound‐closure assay decreased with Cav‐1 overexpression. Wild‐type cells migrated at 14.0 ± 4.9 μm / h (mean ± sd). Transfected cells migrated at 7.0 ± 2.2 μm / h. This was statistically significant by Student's t‐test (p < 0.001). In both wildtype and Cav‐1 transfected BLM melanoma, we were unable to demonstrate altered cell migration rates when unbound CXCL8 was administered over a 24 hour period in RPMI media containing 10% FBS. In summary, Cav‐1 expression in BLM melanoma is associated with an increased N‐cadherin expression, no change in cell division by BrdU assay, and slowed migration by wound‐healing assay. More work will be necessary to elucidate the underlying mechanisms.Support or Funding InformationSupported by the Graduate Program Committee, KCOM

Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells.

Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS.

Uveal melanoma clonogenic response to proton beam irradiation

PurposeThe goal of this study was to compare the cellular clonogenic response to proton beam and X‐ray irradiation of uveal melanoma cell line Mel270 and human melanoma cell line BLM.MethodsBLM line was derived from a skin melanoma metastasis to the lung, and Mel270 is a primary uveal melanoma cell line. Cells were irradiated with 1–5 Gy of X ray (300 kVp Phillips, 1 Gy/min) or proton beam (60 MeV) from Proteus C‐235. After irradiation cells were seeded for the clonogenic assay. Two weeks after seeding the number and type of clones were determined. Three types of clones were found: the largest in size holoclones (with the highest potential to proliferate), medium sized meroclones and the smallest paraclones (low potential to proliferate).ResultsThe total number of clones were similar for both cell lines and both types of irradiation. The RBE values calculated for 37% of survival fraction were 1.11 and 1.13 for Mel270 and BLM cells, respectively. Three types of clones seen in untreated cultures imply the heterogeneity of cellular populations. After irradiation the proportion between the three types of clones was changed: the number of holoclones was drastically lower, and the number of paraclones increased. The number of paraclones was lower after proton bean irradiation in comparison to X‐rays in both cell lines.ConclusionsProton beam and X‐ray irradiation differently modify the proliferation potential of melanoma cells.

Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

BackgroundCutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth.Methods and ResultsProtein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists.ConclusionsOur results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a heterogeneous tumor and that genetic profiling is mandatory for the development of effective personalized therapeutic approaches for melanoma patients.

Epigenetic impacts of ascorbate on human metastatic melanoma cells.

In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.

Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration

The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.

Metastases inhibition and cellular damage in melanoma cells irradiated with proton beam

AbstractPurpose Proton beam therapy has been used in clinic with a great success, however, not many experimental studies has been performed. The goal of our study was to determine the cellular response to low, sublethal doses of proton beam irradiation, in particular DNA damage, cell cycle arrest, changes in expression of proteins, and effect on metastases in vivo.Methods BLM cells were irradiated with 1‐7 Gy of proton beam irradiation. The source of the 58 MeV proton beam was the AIC‐144 cyclotron at Institute of Nuclear Physics, Polish Academy of Sciences, Kraków. Tumors of Bomirski Hamster Melanoma (BHM) implanted into the anterior chamber of the hamster eye grew aggressively and completely filled the anterior chamber within 8‐10 days. Metastases, mainly in the lung, were found in 100% of untreated animals 30 days after enucleation. The protons were accelerated using AIC‐144 isochronous cyclotron, operating at 60 MeV and BHM tumors located in the anterior chamber of the eye were irradiated with 10 Gy, for the depth of 3.88 mm.Results Slow accumulation of damage was observed reflected in slowing of the proliferation rate, and increase in caspases activity with time. The number of cells in G2/M and >2n increased with proton beam dose. Proton beam irradiation caused upregulation of proteins involved in: DNA repair, RNA functioning (i.e. stress granule and P‐bodies components), apoptosis and survival processes and downregulation of enzymes engaged in glycolysis. Of particular interest was heavy downregulation of vimentin (2.4 times), involved in structural integrity of cells and tissues, adhesion and migration, and other processes. Irradiation led to changes in cell migratory properties. Proton beam irradiation caused inhibition of tumor growth by about 10 days and inhibition of metastastic spread.

The helicase domain and C-terminus of human RecQL4 facilitate replication elongation on DNA templates damaged by ionizing radiation

The vertebrate RECQL4 (RECQ4) gene is thought to be the ortholog of budding yeast SLD2. However, RecQL4 contains within its C-terminus a RecQ-like helicase domain, which is absent in Sld2. We established human pre-B lymphocyte Nalm-6 cells, in which the endogenous RECQL4 gene was homozygously targeted such that the entire C-terminus would not be expressed. The RECQL4(ΔC/ΔC) cells behaved like the parental cells during unperturbed DNA replication or after treatment with agents that induce stalling of DNA replication forks, such as hydroxyurea (HU). However, after exposure to ionizing radiation (IR), the RECQL4(ΔC/ΔC) cells exhibited hypersensitivity, inability to complete S phase and prematurely terminated or paused DNA replication forks. Deletion of BLM, a gene that also encodes a RecQ helicase, had the opposite phenotype; an almost wild-type response to IR, but hypersensitivity to HU. Targeting both RECQL4 and BLM resulted in viable cells, which exhibited mostly additive phenotypes compared with those exhibited by the RECQL4(ΔC/ΔC) and the BLM(-/-) cells. We propose that RecQL4 facilitates DNA replication in cells that have been exposed to IR.

Abstract 3464: Melanomas differentially regulate directionally persistent endothelial cell migration

Abstract Introduction: Cutaneous melanoma is the most aggressive form of skin cancer. Although early detection and surgical resection of the primary lesion could improve survival, metastatic melanoma is by large refractory to therapy and predicts poor prognosis. Melanoma progression and metastatic dissemination relies on the process of angiogenesis. The angiogenic process is comprised of a stringent cascade of events ultimately resulting in a new vessel. Pathological (tumor) angiogenesis, however, is mediated by an array of angiogenic modulators produced by the tumor itself. Such tumor-associated modulators arbitrate the enhanced and awry proliferative, survival and migratory responses exhibited by endothelial cells, culminating in unregulated and unimpeded angiogenesis. Project Description: In the current study, we evaluated the angiogenic potential of human melanoma cell lines, based on their ability to regulate directionally persistent endothelial cell (EC) migration. Melanoma conditioned medium (CM) from three human melanoma cell lines (BLM, Mel57, 1F6), differing in their degree of aggressiveness and clinical staging, were used to assess the migratory responses of ECs in a 2-D migration assay, previously established by our group. This assay involves the use of the ‘ring and barrier’ system, wherein the ECs were cultured in a migration ring to generate a confluent area at the periphery and a cell free area in the center. EC migration in response to the tested CMs was monitored real-time, and various migration parameters were determined. Additionally, CM size fractions were tested to delineate the differences observed in migratory responses of the ECs to the tested CMs. Results and Conclusions: There was a clear correlation between EC migration and the degree of aggressiveness of the melanoma cell line used to generate the CM. While the CM of the highly aggressive BLM tumor cells induced a high migratory response in ECs, the CM of the less aggressive 1F6 cells had an inhibitory effect. The 1F6 CM was further size fractionated and it was observed that the factor/s responsible for this inhibition lay in the 10-30 kD range. Gene and protein expression verification helped identify a varied protein expression profile, which are secreted into the CM. Their expression inversely correlated with aggressiveness of the melanoma cell line and ability of the respective CMs to affect EC migration. CM of 1F6 cells silenced with shRNA induced EC migration, while both recombinant protein as well as the CM of BLM cells transfected with protein expressing plasmid mitigated EC migration. Taken together, these results demonstrate that the soluble protein in the CM inhibits the inherent propensity of endothelial cells to undergo directionally persistent migration and that this expression could impede EC melanoma-induced angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3464. doi:10.1158/1538-7445.AM2011-3464

Keratinocytes drive melanoma invasion in a reconstructed skin model

Melanoma progression is a multistep progression from a common melanocytic nevus through the radial growth phase, the invasive vertical growth phase finally leading to metastatic spread into distant organs. Migration and invasion of tumor cells requires secretion of proteases to facilitate remodeling of the extracellular matrix including basement membranes. Here we used a reconstructed skin model to investigate melanoma growth and invasion in vitro. Using this model we show that the dermoepidermal basement membrane prevents the invasion of metastatic melanoma BLM and MV3 cells in the absence of a stratified epidermis. In the reconstructed skin model, matrix metalloproteinase-9, a protease activated early in melanoma development, is secreted by the keratinocytes and subsequently activated by an unknown soluble factor secreted by the melanoma cells. The dynamic interplay between keratinocytes and melanoma cells is further shown by an altered growth pattern of melanoma cells and the finding that a reconstructed epidermis induces invasion. Overall, our findings show that the invasive behavior of melanoma cells is determined by the melanoma cells themselves, but that the interplay between surrounding keratinocytes and the melanoma cells plays an important role in melanoma invasion.