PubMed Health⌕ Search

Biomedical subjects

Douglas Grossman

Publications and source records attributed to Douglas Grossman.

At least 19 recordsLinked to original sources
PubMed2007

Phosphorylation of mitochondrial phospholipid scramblase 3 by protein kinase C-delta induces its activation and facilitates mitochondrial targeting of tBid.

Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.

Apoptosis↗
PubMed2006

Proteolytic cleavage of Livin (ML-IAP) in apoptotic melanoma cells potentially mediated by a non-canonical caspase.

BACKGROUND: Several inhibitor of apoptosis proteins (IAPs) are cleaved during apoptosis. Studies of the melanoma-associated IAP (ML-IAP) Livin, using recombinant molecules, have implicated both caspases 3/7 and the serine protease Omi/HtrA2 in its proteolytic cleavage. OBJECTIVE: To characterize the apoptotic cleavage of Livin in melanocytic cells, and evaluate the role of known proteases. METHODS: We assessed the capacity of a variety of stimuli to induce Livin cleavage in human melanoma cell lines and normal human melanocytes. The role of caspases and Omi was examined using caspase inhibitors and RNAi, respectively. A potential caspase substrate was further examined by site-directed mutagenesis. Deletion mapping was used to identify the cleavage site. RESULTS: Livin cleavage was observed in multiple human melanoma cell lines in response to a variety of apoptotic stimuli (UVB, 4-TBP, cisplatin, TNF, Bax), and not affected by the addition of various protease inhibitors or RNAi-mediated silencing of Omi/HtrA2. Livin cleavage induced by 4-TBP, but not UVB or cisplatin, was blocked by the pan-caspase inhibitor zVAD-fmk. Mutation of Asp52 to Glu in Livin did not affect cleavage, while either mutation of Asp52 to Ala, deletion of Asp52, or deletion of the adjacent region (residues 53-61) abrogated cleavage. CONCLUSION: Livin cleavage, induced by multiple apoptotic stimuli in melanoma cells, likely occurs in an Omi-independent fashion at residue 52 within its potential caspase substrate (DHVD52). However, relative insensitivity of the apoptotic cleavage to zVAD-fmk, or Asp52 to Glu mutation, suggests the involvement of a non-canonical caspase.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Activation of dual apoptotic pathways in human melanocytes and protection by survivin.

Apoptosis resistance in melanoma is a primary cause of treatment failure. Apoptotic pathways in melanocytes, from which melanoma arises, are poorly characterized. Human melanocytes were susceptible to apoptosis following exposure to UV radiation (UVB, 24-48 hours), 4-tert-butylphenol (4-TBP, 1-4 hours), and cisplatin (24-48 hours). These responses were associated with Bid cleavage, caspase activation (caspases 3, 8, and 9), mitochondrial depolarization and release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor (AIF), but not endonuclease G. The apoptotic responses and AIF release were caspase-independent, as they were not blocked by zVal-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk). While RNA interference-mediated knockdown of AIF protected melanocytes against apoptosis induced by serum withdrawal, apoptotic responses to UVB, cisplatin, and 4-TBP were not compromised by AIF knockdown, even in the presence of zVAD-fmk. Finally, adenoviral-mediated expression of Survivin, an inhibitor of apoptosis expressed in melanoma but not melanocytes, protected melanocytes against UVB-induced apoptosis. Survivin expression in melanocytes partially blocked caspase activation and release of mitochondrial release of AIF, cytochrome c, and Smac induced by UVB. These data indicate that multiple stimuli can activate both caspase-dependent and caspase-independent apoptotic pathways in melanocytes, and that endogenous expression of Survivin in melanoma may contribute to apoptosis resistance by multiple mechanisms.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Keratinocyte apoptosis in epidermal development and disease.

Keratinocyte (KC) apoptosis plays a critical role in regulating epidermal development and restraining carcinogenesis. Apoptosis balances proliferation to maintain epidermal thickness, contributes to stratum corneum formation and may eliminate pre-malignant cells. Apart from the normal developmental program, KC apoptosis can be triggered by UV light and other stimuli. Dysfunctional apoptosis occurs in some skin diseases, such as psoriasis and skin cancer. Here we review the current state of knowledge of KC apoptosis, with particular focus on apoptotic signaling pathways and molecular mechanisms of apoptosis control, and discuss new insights into the complex role of apoptosis in skin carcinogenesis that are emerging from mouse models.

Animals↗
PubMed2006

Melanoma genetics: a review of genetic factors and clinical phenotypes in familial melanoma.

PURPOSE OF REVIEW: The clinical phenotypes of familial melanoma syndromes and genetic and environmental interactions are reviewed to summarize the current status of the field and to identify gaps in molecular and clinical investigations. RECENT FINDINGS: The familial melanoma syndromes are associated with germline mutations in three highly penetrant gene products: p16, alternate reading frame, and cyclin-dependent kinase 4. Certain variants in a low-penetrance gene, MC1R, the melanocortin 1 receptor gene, increase melanoma risk to a lesser extent and act as a genetic modifier when cosegregating with a deleterious p16 gene. The penetrance of these melanoma-predisposing genes is largely influenced by ultraviolet exposure across geographic latitude. Yet cumulative studies are conflicting on whether ultraviolet radiation, including sunburns, early childhood and adolescent sun exposure, and chronic exposure, increases melanoma risk in familial melanoma. To date, the clinical phenotypes of increased number of atypical nevi and nevi body distribution are independent risk factors for melanoma risk, regardless of family history. The atypical mole syndrome cannot reliably predict melanoma germline mutations but increases melanoma risk in p16 mutation carriers. Familial melanoma patients develop melanomas earlier and are prone to developing multiple primary melanomas. Other than these two differences, familial and sporadic melanoma share similar histopathology, prognostic factors, and survival rates. SUMMARY: Familial melanoma is an excellent human model system for the investigation of melanoma. Understanding genotype-phenotype and environmental relationships in familial melanoma will likely lead to improved understanding of pathogenesis for all melanoma patients.

Cyclin-Dependent Kinase 4↗
PubMed2005

Colonization of adjacent stem cell compartments by mutant keratinocytes.

A key step in cancer development is clonal expansion. The increased number of mutant cells allows a clinical phenotype and increases the probability that one of the cells will be mutated in an additional gene. For skin cancer, observations on p53-mutant keratinocyte clones in epidermal sheets of UVB-irradiated mice reveal that mutant stem cells are normally restrained within their stem cell compartment. Chronic UVB exposure drives clonal expansion by a non-mutational mechanism, and this mechanism allows mutant stem cells to escape from their own stem cell compartment and colonize adjacent compartments. In the absence of escape, cells proliferate without occupying additional territory. The non-mutational clonal expansion mechanism appears to be UVB-induced apoptosis, which deletes DNA-damaged cells in unmutated stem cell compartments but will preferentially spare death-resistant p53-mutant cells. An additional source of apoptotic selection pressure may come from UV-irradiated melanin.

Animals↗
PubMed2005

Proliferation, apoptosis, and survivin expression in a spectrum of melanocytic nevi.

BACKGROUND: Apoptosis is important for maintenance of tissue homeostasis and often dysregulated in cutaneous neoplasms. The apoptosis inhibitor survivin is expressed in melanoma and non-melanoma skin cancers and benign keratinocytic lesions. Its expression has not been studied in melanocytic nevi. OBJECTIVE: We determined the expression pattern of survivin in benign melanocytic nevi in comparison to markers of proliferation and apoptosis. METHODS: Six cases of each of the following melanocytic nevi were retrieved from a dermatopathology archive: compound dysplastic nevus, intradermal nevus, compound nevus, neurotized intradermal nevus, and Spitz nevus. Survivin expression was evaluated by in situ hybridization. Apoptotic and proliferation indices were calculated by counting immunoreactive cells in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and proliferating cell nuclear antigen immunostained sections, respectively. RESULTS: All nevi, regardless of histologic type, expressed survivin. Compound melanocytic lesions expressed survivin in both epidermal and dermal compartments. The apoptotic rate was low for dysplastic, compound, and Spitz nevi, and apoptotic cells were not identified in any neurotized nevus. The proliferative index was highest for Spitz nevi, while all other nevi demonstrated rare positive cells. CONCLUSIONS: Survivin is consistently expressed in benign melanocytic lesions, while apoptotic cells are rarely identified, suggesting the dysregulation of apoptotic pathways with the accumulation of cells in these neoplasms.

Adolescent↗
PubMed2004

UVB-induced apoptosis drives clonal expansion during skin tumor development.

The mechanism by which a single mutant cell clonally expands is usually assumed to involve an additional mutation in a cell cycle regulatory gene. An alternative mechanism for driving clonal expansion is apoptosis, which might create vacant stem cell compartments that can be repopulated by mutant cells. This model predicts that in a mouse with reduced apoptotic capacity (i) more mutated cells will appear initially but (ii) these cells will expand into clones more slowly than in wild-type animals. To test this hypothesis for ultraviolet B (UVB)-induced skin carcinogenesis, we examined UVB-induced p53 mutant clones and tumors in a transgenic (Tg) mouse (K14-Survivin) with skin-specific expression of the apoptosis inhibitor Survivin. To limit the effects of Survivin on apoptosis, without affecting epidermal proliferation or differentiation, we used Survivin expression levels and UVB doses that resulted in a 2-fold reduction in keratinocyte apoptosis. After 5 weeks of chronic UVB irradiation, newly created p53 mutant keratinocyte clones (indicative of initial mutation frequency) were 1.4-fold more frequent in K14-Survivin mice (P = 4 x 10(-6)). As predicted, this effect was reversed for clones growing by clonal expansion, which were rarer in Tg skin by 1.7-fold (P = 0.047). At 10 weeks large expanding Tg clones were rarer by a magnitude approaching the apoptosis differential (approximately 2-fold, P = 4 x 10(-5)). Survivin expression also retarded clonal expansion at later stages of tumor development. By 20 weeks 95% of animals carried tumors (primarily papillomas), which were 1.6-fold rarer in apoptosis-defective Tg mice (P = 0.03). In contrast, the rate of tumors attaining large size (> or =3 mm, P = 0.048) and converting to carcinoma was increased approximately 2-fold in Tg mice. Thus, Survivin-regulated apoptosis appears to suppress two stages that involve new mutations, initiation and malignant conversion, yet drives clonal expansion of existing p53 mutant cells.

Animals↗
PubMed2004

Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells.

The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key antiapoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis.

Active Transport, Cell Nucleus↗
PubMed2004

Vulvar melanoma: diffuse melanosis and metastasis to the placenta.

Mucocutaneous melanoma, including vulvar melanoma, is rare and has a worse prognosis and higher recurrence rate than traditional cutaneous melanoma. Diffuse cutaneous melanosis is another rare clinical presentation of metastatic melanoma. It is essential for dermatologists to be alerted to rare presentations of melanoma, to facilitate early detection. We present the first case to our knowledge of metastatic vulvar melanoma with diffuse cutaneous melanosis in a pregnant young woman. Despite the occurrence of placental metastasis, a healthy, unaffected baby was born. This case exemplifies the aggressiveness of vulvar melanoma. The genitalia should be included in routine total body skin examinations. Pregnant women with generalized melanosis may be at increased risk for placental metastasis of melanoma. Pregnancy does not alter the incidence or prognosis of melanoma; however, patients with a poor prognosis or high recurrence risk should be informed of potential pregnancy complications associated with melanoma recurrence or metastasis.

Adult↗
PubMed2004

Proliferation, apoptosis, and survivin expression in keratinocytic neoplasms and hyperplasias.

The dysregulation of apoptosis occurs in many cutaneous disease states. Several apoptosis inhibitors have been shown elevated in neoplasms and in some inflammatory conditions, but their relation to proliferative and apoptotic states has not been defined. We examined the expression of the apoptosis inhibitor survivin in a panel of keratinocytic neoplasms and hyperproliferative skin lesions using both immunohistochemistry and a newly developed in situ hybridization technique. Proliferation and apoptotic indices were also assessed by immunohistochemical staining for proliferating cell nuclear antigen and TUNEL, respectively. We found the highest rate of proliferation in verrucae and psoriasis followed by actinic keratosis, squamous and basal cell carcinoma, lichen simplex chronicus, and seborrheic keratosis; all were significantly (P < 0.05) higher than normal skin. Apoptotic rate was increased in squamous (P = 0.05) and basal cell carcinoma (P = 0.03), but not significantly different from normal skin in the other lesions tested. Survivin expression was seen in most neoplasms and hyperproliferative lesions, but not normal skin. Survivin expression was often restricted to the upper third of the epidermis in psoriasis and lichen simplex chronicus, whereas all the other lesions stained diffusely. Survivin expression appears to be a consistent feature of keratinocytic neoplasms and hyperproliferative lesions and may contribute to the formation of epidermal hyperplasia seen in all of these disease states.

Antigens, Neoplasm↗
PubMed2004

Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine.

It remains unclear to what extent drugs targeting transcriptional repressor complexes affect global gene expression in cells derived from target and nontarget human tissues. To address this question, we used genome-wide expression analysis using microarrays to analyze the response of three tumor and one normal epithelial cell line to treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). Notably, we found that 5-aza-CdR treatment induced a limited number of genes (mean, 0.67%; range, 0.17-1.8% of 25,940 genes screened) in each cell line tested. The majority of the gene expression changes that followed 5-aza-CdR treatment were conserved in tumor and normal cells, including genes that function in cell proliferation, differentiation, immune presentation, and cytokine signaling. In contrast, 5-aza-CdR treatment induced the expression of cancer-testis class tumor antigens only in tumor cell lines. To explain this tissue-specific response, we analyzed the mechanism of transcriptional regulation of the prototype member of this tumor antigen gene family, MAGE-1. Taken from our analysis of MAGE-1 gene regulation, we propose that 5-aza-CdR-mediated gene activation has two distinct requirements: 1) the reversal of promoter hypermethylation, and 2) the presence of transcriptional activators competent for the activation of hypomethylated target promoters. This latter requirement for gene activation by 5-aza-CdR is probably mediated by sequence-specific transcription factors and may account for the limited number of human genes induced by 5-aza-CdR treatment. This revised model for gene activation by 5-aza-CdR has important implications for the use of DNA methyltransferase inhibitors in clinical settings.

Antigens, Neoplasm↗
PubMed2003

Metastatic melanoma in pregnancy: risk of transplacental metastases in the infant.

PURPOSE: Although metastases to the fetus via the placenta are rare, melanoma is the most common culprit. When it occurs, maternally derived melanoma metastasis in the infant is almost invariably fatal. PATIENTS AND METHODS: This article reviews current guidelines for placental evaluation in pregnant women with metastatic melanoma and presents surveillance recommendations for their infants. Comprehensive literature reviews were performed on melanoma in pregnancy and melanoma metastasis to the placenta and fetus. The use of interferon alfa in the pediatric population was also reviewed. A comprehensive search of the MEDLINE database (1966 to 2002) was performed. Articles were reviewed and additional references were obtained from the bibliographies. Translation of non-English articles was performed, and authors of previous publications were contacted. RESULTS: Eighty-seven patients with placental or fetal metastasis were identified. Twenty-seven occurrences were attributed to melanoma (31%). The fetus was affected in six of 27 melanoma patients (22%), with five of six infants dying of disease. The use of high-dose interferon alfa adjuvant therapy in pediatric patients has not been reported. CONCLUSION: The placentas of women with known or suspected metastatic melanoma should be carefully examined grossly and histologically by pathologists. With placental involvement, fetal risk of melanoma metastasis is approximately 22%. Neonates delivered with concomitant placental involvement should be considered a high-risk population. The risk-benefit ratio of adjuvant treatment for a potentially affected infant should be carefully weighed.

Female↗
PubMed2003

Survivin expression in mouse skin prevents papilloma regression and promotes chemical-induced tumor progression.

Induction of cutaneous squamous cell carcinoma (SCC) in mice, by topical chemical [9,10-dimethylbenzanthracene (DMBA) and phorbol 12-myristate 13-acetate (PMA)] application, is a multistep process involving papilloma formation and progression to carcinoma. We have generated a transgenic (Tg) mouse [keratin-14 (K14)-survivin] with skin expression of survivin, an inhibitor of apoptosis expressed in most human skin cancers and premalignant lesions. K14-survivin mice were resistant to DMBA-induced keratinocyte apoptosis. To investigate the role of survivin and apoptosis in cutaneous carcinogenesis, mice were treated once topically with DMBA followed by twice weekly with PMA for 32 weeks. Surprisingly, tumor formation was less frequent (31% versus 43%) and significantly delayed (P = 0.01) in K14-survivin mice compared with non-Tg littermates. On the other hand, papilloma regression was not observed in Tg mice, whereas 20% of papillomas regressed in non-Tgs; one SCC was generated in Tg mice, whereas none were seen in non-Tgs. To increase tumor formation and SCC in particular, a second experiment was performed with mice on a p53+/- background. Again, DMBA/PMA-induced tumor formation was less (71% versus 89%) and significantly delayed (P = 0.02) in K14-survivin p53+/- animals compared with p53+/- non-Tgs. Papilloma regression was also not observed in Tg p53+/- mice, whereas 10% of papillomas regressed in p53+/- non-Tgs. The rate of papilloma progression to SCC was 21% in Tg p53+/- mice compared with 12% in p53+/- non-Tgs. Papillomas did not reveal significant differences in mitotic or apoptotic indices. Survivin expression was detected in all of the tumors. These results indicate that despite a paradoxical negative effect on tumor formation, survivin expression prevents papilloma regression and promotes conversion to SCC, consistent with its expression in most skin cancers and their precursors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2003

Apoptosis regulators and responses in human melanocytic and keratinocytic cells.

Apoptosis in keratinocytes is required for epidermal turnover, stratum corneum formation, and removal of ultraviolet-damaged premalignant cells. Its role in melanocyte homeostasis and transformation, on the other hand, has not been defined, although apoptosis resistance is a commonly recognized feature of melanoma. We examined the expression of apoptosis regulators in melanocytes, keratinocytes, melanoma, and HaCat cells. Melanocytic cells expressed relatively high levels of Bcl-2, Bcl-X(L), Mcl-1, C-IAP-1, C-IAP-2, XIAP, Livin, and Apaf-1. The only apoptotic regulator that was differentially expressed in melanoma cells and not melanocytes was Survivin, whereas Bax was expressed in melanocytes but not in most melanoma lines. Keratinocytic cells, on the other hand, expressed high levels of FLIP and were relatively deficient in Bcl-2 family proteins. Levels of p53 were highest in HaCat cells and some of the melanoma lines, and barely detectable in melanocytes and keratinocytes. Next, susceptibility of these cells types to apoptosis induced by ultraviolet B, the tyrosine analog 4-tert-butylphenol, and cytotoxic drugs was examined. Melanocytes were relatively resistant to ultraviolet B, whereas keratinocytes were unresponsive to 4-tert-butylphenol. Melanocytes and keratinocytes were generally less susceptible than melanoma lines and HaCat cells to etoposide, cisplatin, and staurosporine. Induction of apoptosis in these cell types was generally associated with decreased levels of Mcl-1, XIAP, and Livin, and increased levels of p53, whereas levels of other apoptotic regulators were unaltered. These results provide insights into the potential roles of apoptosis in the function and transformation of epidermal melanocytes and keratinocytes.

Apoptosis↗
PubMed2003

Novel application of a fibrin cell block method to evaluate melanocytic cell populations.

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.

Adult↗