CD133 Stimulates Cell Proliferation via the Upregulation of Amphiregulin in Melanoma.

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.

Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers.

The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2ß1 integrin, which is present only on basal and stem cells. The presence of ß1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.

Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells.

Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.

Hypopigmented burn hypertrophic scar contains melanocytes that can be signaled to re-pigment by synthetic alpha-melanocyte stimulating hormone in vitro.

There are limited treatments for dyschromia in burn hypertrophic scars (HTSs). Initial work in Duroc pig models showed that regions of scar that are light or dark have equal numbers of melanocytes. This study aims to confirm melanocyte presence in regions of hypo- and hyper-pigmentation in an animal model and patient samples. In a Duroc pig model, melanocyte presence was confirmed using en face staining. Patients with dyschromic HTSs had demographic, injury details, and melanin indices collected. Punch biopsies were taken of regions of hyper-, hypo-, or normally pigmented scar and skin. Biopsies were processed to obtain epidermal sheets (ESs). A subset of ESs were en face stained with melanocyte marker, S100ß. Melanocytes were isolated from a different subset. Melanocytes were treated with NDP α-MSH, a pigmentation stimulator. mRNA was isolated from cells, and was used to evaluate gene expression of melanin-synthetic genes. In patient and pig scars, regions of hyper-, hypo-, and normal pigmentation had significantly different melanin indices. S100ß en face staining showed that regions of hyper- and hypo-pigmentation contained the same number of melanocytes, but these cells had different dendricity/activity. Treatment of hypo-pigmented melanocytes with NDP α-MSH produced melanin by microscopy. Melanin-synthetic genes were upregulated in treated cells over controls. While traditionally it may be thought that hypopigmented regions of burn HTS display this phenotype because of the absence of pigment-producing cells, these data show that inactive melanocytes are present in these scar regions. By treating with a pigment stimulator, cells can be induced to re-pigment.

Inorganic polyphosphate in platelet rich plasma accelerates re-epithelialization in vitro and in vivo.

Wound healing requires well-coordinated events including hemostasis, inflammation, proliferation, and remodeling. Delays in any of these stages leads to chronic wounds, infections, and hypertrophic scarring. Burn wounds are particularly problematic, and may require intervention to ensure timely progression to reduce morbidity and mortality. To accelerate burn wound healing, Platelet-Rich Plasma (PRP)1 can be of value, since platelets release growth factor proteins and inorganic polyphosphates (polyP) that may be integral to wound healing. We used polyP-depleted keratinocyte (HaCaT) and fibroblast cell culture models to determine cell proliferation and scratch-wound repair to determine if polyP, platelet lysate, or combined treatment could accelerate wound healing. While polyP and PRP significantly reduced the open scratch-wound area in fibroblasts and keratinocytes, polyP had no effect on keratinocyte or fibroblast proliferation. PRP was also evaluated as a treatment in a murine model of full thickness wound healing in vivo, including a treatment in which PRP was supplemented with purified polyP. PRP induced significantly more rapid re-epithelialization by Day 3. Pure polyP enhanced the effects of PRP on epithelial tongues, which were significantly elongated in the PRP + high-dose polyP treatment groups compared to PRP alone. Thus, PRP and polyP may serve as an effective therapeutic combination for treating wounds.

Promoter Methylation Status in Pro-opiomelanocortin Does Not Contribute to Dyspigmentation in Hypertrophic Scar.

Burn injuries frequently result in hypertrophic scars (HTSs), specifically when excision and grafting are delayed due to limited resources or patient complications. In patient populations with dark baseline pigmentation, one symptom of HTS that often occurs is dyspigmentation. The mechanism behind dyspigmentation has not been explored, and, as such, prevention and treatment strategies for this morbidity are lacking. The mechanism by which cells make pigment is controlled at the apex of the pathway by pro-opiomelanocortin (POMC), which is cleaved to its products alpha-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropin hormone (ACTH). α-MSH and ACTH secreted by keratinocytes bind to melanocortin 1 receptor (MC1R), expressed on melanocytes, to initiate melanogenesis. POMC protein expression is upregulated in hyperpigmented scar compared to hypopigmented scar by an unknown mechanism in a Duroc pig model of HTS. POMC RNA levels, as well as the POMC gene promoter methylation status were investigated as a possible mechanism. DNA was isolated from biopsies obtained from distinct areas of hyper- or hypopigmented scar and normal skin. DNA was bisulfite-converted, and amplified using two sets of primers to observe methylation patterns in two different CpG islands near the POMC promoter. Amplicons were then sequenced and methylation patterns were evaluated. POMC gene expression was significantly downregulated in hypopigmented scar compared to normal skin, consistent with previously reported protein expression levels. There were significant changes in methylation of the POMC promoter; however, none that would account for the development of hyper- or hypopigmentation. Future work will focus on other areas of POMC transcriptional regulation.

CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis.

CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.

CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition.

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibition in vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as "melanoma initiating cells," resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

Reactive Oxygen Species Scavenging Potential Contributes to Hypertrophic Scar Formation.

BACKGROUND: Reactive oxygen species (ROS) can damage macromolecules if not appropriately neutralized by ROS scavengers. The balance between ROS and ROS scavengers is essential to prevent the accumulation of damage in healthy tissues. This balance is perturbed in hypertrophic scar (HTS). MATERIALS AND METHODS: Full-thickness wounds were created on the flanks of Duroc pigs at day 0 that developed into HTS (n = 4). Wounds and HTSs were biopsied weekly for 135 d. Total transcriptome microarrays were conducted with focused ROS scavenger analysis. Confirmatory quantitative reverse transcription polymerase chain reaction and immunofluorescence of ROS scavengers: superoxide dismutase 1, microsomal glutathione S-transferase 1, and peroxiredoxin 6 were performed throughout wound healing and HTS development. RESULTS: Total transcriptome microarray analysis identified over 25 ROS scavenger genes that were significantly downregulated in HTS at all time points compared with basal level controls (BL) (FDR<0.01; fold change > or <2). Ingenuity pathway analysis identified multiple ROS scavenging pathways involved in HTS (P < 0.01). Quantitative reverse transcription polymerase chain reaction of representative scavengers confirmed and expanded this finding to the initial phases of wound healing (P < 0.05, n = 4). The protein products of the genes were lower in wound and HTS tissues compared with BL. CONCLUSIONS: A balance between ROS production and scavenging must be maintained for normal wound healing, which is perturbed in wounds that heal to form HTSs. We postulate that endogenous scavengers can be administered as a prophylactic or post-treatment to rebalance ROS and attenuate symptoms of scar.

Pigmentation Diathesis of Hypertrophic Scar: An Examination of Known Signaling Pathways to Elucidate the Molecular Pathophysiology of Injury-Related Dyschromia.

Hypertrophic scar (HTS) occurs frequently after burn injury. Treatments for some aspects of scar morbidity exist, however, dyspigmentation treatments are lacking due to limited knowledge about why scars display dyschromic phenotypes. Full thickness wounds were created on duroc pigs that healed to form dyschromic HTS. HTS biopsies and primary cell cultures were then used to study pigmentation signaling. Biopsies of areas of both pigment types were taken for analysis. At the end of the experiment, melanocyte-keratinocyte cocultures were established from areas of differential pigmentation. Heterogeneously dyspigmented scars formed with regions of hyperpigmentation and hypopigmentation. Melanocytes were present in both pigment types measured by S100ß quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining, and visualized by dendritic cell presence in primary cultures. P53 expression was not different between the two pigment types. Hyperpigmented scars had upregulated levels of proopiomelanocortin (POMC), adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), stem cell factor (SCF), and c-KIT and melanocortin 1 receptors (MC1R) compared to hypopigmented regions. Many genes involved in dyspigmentation were differentially regulated by microarray analysis including MITF, TYR, TYRP1, and DCT. MiTF expression was not different upon further exploration, but TYR, TYRP1, and DCT were upregulated in intact biopsies measured by qRT-PCR and confirmed by immunostaining. This is the first work to confirm the presence of melanocytes in hypopigmented scar using qRT-PCR and primary cell culture. An understanding of the initial steps in dyspigmentation signaling, as well as the downstream effects of these signals, will inform treatment options for patients with scars and provide insight to where pharmacotherapy may be directed.

Treatment Strategies for Hypopigmentation in the Context of Burn Hypertrophic Scars.

Dyspigmentation in burn scars can contribute to the development of psychosocial complications after injury and can be detrimental to social reintegration and quality of life for burn survivors. Although treatments for skin lightening to treat hyperpigmentation have been well reviewed in the literature, skin-darkening strategies to treat hypopigmentation have not. The following potential treatment options in the context of burn hypertrophic scar will be discussed: use of the melanocyte-keratinocyte transplantation procedure, use of ectopic synthetic analogues of alpha-melanocyte stimulating hormone to initiate melanogenesis, and use of FK506 to induce melanogenesis. A proposed future direction of research in laser-assisted drug delivery of inducers of local melanin production, with the hope of developing a targeted, effective approach to dyspigmentation in hypertrophic scar is also discussed.

The repurposed anthelmintic mebendazole in combination with trametinib suppresses refractory NRASQ61K melanoma.

Structure-based drug repositioning in addition to random chemical screening is now a viable route to rapid drug development. Proteochemometric computational methods coupled with kinase assays showed that mebendazole (MBZ) binds and inhibits kinases important in cancer, especially both BRAFWT and BRAFV600E. We find that MBZ synergizes with the MEK inhibitor trametinib to inhibit growth of BRAFWT-NRASQ61K melanoma cells in culture and in xenografts, and markedly decreased MEK and ERK phosphorylation. Reverse Phase Protein Array (RPPA) and immunoblot analyses show that both trametinib and MBZ inhibit the MAPK pathway, and cluster analysis revealed a protein cluster showing strong MBZ+trametinib - inhibited phosphorylation of MEK and ERK within 10 minutes, and its direct and indirect downstream targets related to stress response and translation, including ElK1 and RSKs within 30 minutes. Downstream ERK targets for cell cycle, including cMYC, were down-regulated, consistent with S- phase suppression by MBZ+trametinib, while apoptosis markers, including cleaved caspase-3, cleaved PARP and a sub-G1 population, were all increased with time. These data suggest that MBZ, a well-tolerated off-patent approved drug, should be considered as a therapeutic option in combination with trametinib, for patients with NRASQ61mut or other non-V600E BRAF mutant melanomas.

Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.

Inhibitor of differentiation-4 (Id4) stimulates pigmentation in melanoma leading to histiocyte infiltration.

TGF-ß and the inhibitors of differentiation (Id) are linked. Smad7 and other TGF-ß inhibitors can potently suppress melanomagenesis; however, little work examining Ids has been reported in melanoma, particularly for Id4. Here, we report that Id4, but not Id2 or Id3 expression, surprisingly, activated robust melanin production in xenografts of previously amelanotic (lacking pigment) 1205Lu/Smad7 (S7) cells. Fontana-Masson stain and de-novo expression of MART-1 and tyrosinase proteins confirmed melanin production. Additionally, pigment-laden CD163+ mouse histiocytes with areas of extensive necrosis were found throughout S7/Id4 tumors, but not in parental 1205Lu, S7/Id2 or S7Id3-derived tumors. Mechanistic investigation revealed increased nuclear M-microphthalmia-associated transcription factor (MITF) and MART-1 promoter activation following Id4 expression in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanin synthesis. In human tumors, melanin colocalized with Id4 expression establishing a correlation. Current chemotherapeutics for melanoma are only marginally effective. Immunotherapy provides the most promise, yet the role of innate immunity is poorly understood. Here, TGF-ß suppression followed by Id4 expression results in extensive melanin synthesis and robust histiocyte recruitment following tumorigenesis, a novel role for Id4. Our results suggest that TGF-ß suppression coupled with pigment overproduction triggers an innate immune response resulting in tumor necrosis.

Id2, Id3 and Id4 overcome a Smad7-mediated block in tumorigenesis, generating TGF-ß-independent melanoma.

The role for the inhibitors of differentiation (Ids) proteins in melanomagenesis has been poorly explored. In other cell types, Ids have been shown to contribute to cell proliferation, migration and angiogenesis and, along with a number of other genes, are direct downstream targets of the transforming growth factor (TGF)-ß pathway. Expression of Smad7, which suppress TGF-ß signaling, or synthetic TGF-ß inhibitors, was shown to potently suppress melanomagenesis. We found that endogenous Id2, Id3 and Id4 expression was elevated in 1205Lu versus 1205Lu cells constitutively expressing Smad7, indicating Ids may play a role in melanomagenesis. Therefore, the effects of Tet-inducible expression of Id2, Id3 or Id4 along with Smad7 in TGF-ß-dependent 1205Lu human melanoma cells were explored in vitro and in vivo. 1205Lu cells formed subcutaneous tumors in athymic mice, whereas cells expressing Smad7 failed to form tumors. However, 1205Lu cells expressing Smad7 along with doxycycline-induced Id2, Id3 or Id4 were able to overcome the potent tumorigenic block mediated by S7, to varying degrees. Conversely, Id small interfering RNA knockdown suppressed anchorage-independent growth of melanoma. Histology of tumors from 1205Lu cells expressing Smad7 + Id4 revealed an average of 31% necrosis, compared with 5.2% in tumors from 1205Lu with vector only. Downstream, Ids suppressed cyclin-dependent kinase inhibitors, and re-upregulated invasion and metastasis-related genes matrix metalloproteinase 2 (MMP2), MMP9, CXCR4 and osteopontin, shown previously to be downregulated in response to Smad7. This study shows that Id2, Id3 and Id4 are each able to overcome TGF-ß dependence, and establish a role for Ids as key mediators of TGF-ß melanomagenesis.

Clonal dominance of CD133+ subset population as risk factor in tumor progression and disease recurrence of human cutaneous melanoma.

Chemotherapeutic refractoriness of advanced cutaneous melanoma may be linked with melanoma-initiating cells, also known as melanoma stem cells. This study aimed to determine relative risk of clonal dominance of the CD133+ phenotype in tissues from melanoma patients with different clinical outcomes that could be applied to early diagnosis, prognosis or disease monitoring. Significant overexpression of CD133 (p<0.02) was observed by immunohistochemical staining in tissues from patients with recurrent disease versus those without disease recurrence. Relative risk analysis between these two groups suggested that the patients with recurrence or metastatic lesion had a greater than 2-fold overexpression of CD133. In addition, immunodetectable CD133 corroborated with upregulation of CD133 RNA levels (14- to 30-fold) as assessed by quantitative real-time reverse transcription-PCR (qRT-PCR) comparison of melanoma cell lines derived from patients with poor clinical outcomes and short overall survival (<10 months), vs. those derived from patients with good clinical outcomes and longer overall survival (>24 months). Further, cells derived from patients, and MACS-sorted according to their CD133 status retained their CD133-positivity (>95%) or CD133-negativity (>95%) for more than 8 passages in culture. CD133+ cells could repopulate and form tumors (p<0.03) in athymic NCr-nu/nu mice within 8 weeks while no tumors were observed with CD133- phenotype (up to 200,000 cells). Taken together, the study demonstrates, for the first time, that there exists a clonal dominance of a CD133+ population within the hierarchy of cells in cutaneous tissues from patients that have undergone successive progressive stages of melanoma, from primary to metastatic lesions. CD133, thus, provides a predictive marker of disease as well as a potential therapeutic target of high-risk melanoma.

Purification and characterization of poly(ADP-ribosyl)ated DNA replication/repair complexes.

PARP-1, the best studied isoform and most abundantly expressed member of the PARP family of 18 proteins, catalyzes the poly(ADP-ribosyl)ation (PARylation) of various nuclear proteins and play key roles in DNA repair, genome maintenance, DNA replication, recombination, apoptosis, gene expression, and regulation of chromatin function. PARylation modulates the functions of target proteins, mainly PARP-1 itself. A multifunctional enzyme, PARP-1 has been localized within DNA replication, repair, recombination, and transcription complexes, and modifies and regulates the functions of specific components of these complexes. PARylation can regulate the activities of replicative enzymes, such as DNA polymerases α, δ, and ε, topo I and II, primase, RPA, and PCNA in isolated enzymes or within DNA replication complexes (DNA synthesome). PARP-1 and PARylation may (1) play dual roles in nuclear processes, depending on the levels of the substrate NAD and the presence of PARP-activating DNA breaks, (2) recruit acceptor proteins to certain sites or complexes through direct association or through binding to PAR and PAR-binding proteins, and (3) alters the nucleosomal structure of DNA by PARylation of nucleosomal proteins, such as histone H1 to destabilize higher order chromatin structures and promote access of DNA repair and replication enzymes as well as transcription factors to these sites. Here, we describe biochemical approaches that have been utilized in our laboratory for the purification and characterization of PARylated DNA replicative complexes. These methods can be modified for the purification of complexes involved in other nuclear processes. This chapter also briefly discusses current methods by which new PARylated complexes are being identified and studied. Identification, evaluation, and characterization of new complexes could aid in the elucidation of the molecular mechanisms by which PARylation and PARP mediates its pleiotropic roles in various nuclear processes.

Proteomic analysis of pathways involved in estrogen-induced growth and apoptosis of breast cancer cells.

BACKGROUND: Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation. CONCLUSIONS: G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen.

A review of the local pathophysiologic bases of burn wound progression.

Burn wound progression refers to the phenomenon of continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult. A multitude of chemical and mechanical factors contribute to the local pathophysiologic process of burn wound progression. Prolonged inflammation results in an accumulation of cytotoxic cytokines and free radicals, along with neutrophil plugging of dermal venules. Increased vascular permeability and augmentations of interstitial hydrostatic pressure lead to edema with vascular congestion. Hypercoagulability with thrombosis further impairs blood flow, while oxidative stress damages endothelial cells and compromises vascular patency. A number of studies have investigated the utility of various agents in modulating these mechanisms of burn wound progression. However, as many of studies have used animal models of burn injury, often with administration of therapy preburn, obscuring the clinical applicability of the results to burn patients is of questionable benefit. An understanding of the complex, interrelated mediators of burn wound progression and their ultimate point of convergence in effecting tissue necrosis­cell apoptosis or oncosis­will allow for the future development of therapeutic interventions.