Micropthalmia Transcription Factor Research Articles

Histone deacetylase inhibitor Vorinostat (SAHA) suppresses micropthalmia transcription factor expression and induces cell death in nevocytes from large/giant congenital melanocytic nevi.

Large/giant congenital nevi (L/GCMN) are benign neoplasms of the melanocytic neural crest lineage covering extensive areas of skin presenting risk for melanoma. Surgical resection often leads to scarring and trauma. Histone deacetylase inhibitors (iHDACs) as topical therapeutic agents may prove beneficial as an alternative/adjunct to surgery in this disease. Here we describe the effect of in vitro treatment of iHDACs drugs on primary nevocytes isolated from L/GCMN patients. Micropthalmia transcription factor (MITF) expression in L/GCMN patients' lesions was detected by immunohistochemistry, in cultured nevocytes by immunofluorescence, immunoblot and quantitative polymerase chain reaction. Cellular senescence was detected by SA-ß galactosidase activity. Markers for melanocytic differentiation were evaluated by immunoblot analysis and extracted melanin content was estimated spectrophotometrically. Cell death was measured by lactate dehydrogenase (LDH) assay and necrosis confirmed by polymerase (PARP) cleavage and acridine orange staining of the nuclei. MITF was expressed ubiquitously in nevocytes and melanocytes in patients' lesions. In culture, iHDAC treatment suppressed MITF protein and mRNA expression resulting in a senescent-like phenotype with positive ß-galactosidase staining, progressing to necrotic cell death as evidenced by increased LDH activity, appearance of cleaved PARP and necrotic nuclei. This is the first report showing evidence of iHDACs-induced MITF suppression in congenital nevocytes in vitro leading to a morphologic change with positive ß-galactosidase staining, followed by necrotic cell death in nevocytes, indicating that iHDAC drugs could be valuable therapeutic agents for treatment of L/GCMN lesions.

Abstract 3109: Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P<0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P<0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner. Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3109.

Abstract 4475: Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathway resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we sought to study the biology underlying oncogenesis in tRCC involving TFE3 fusion with genes associated with pre-mRNA splicing factor machinery; NONO, SFPQ and PRCC. Methods: To investigate the biology of different TFE3 fusion proteins, oncogenic phenotypes and oncogenic signaling were compared among cells with distinct endogenous expression of TFE3 fusions such as SFPQ-TFE3, NONO-TFE3, PRCC-TFE3. Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. TFE3 fusion transcripts were identified with RT-PCR. TFE3 fusion preferential DNA binding was assessed with ChIP-sequencing. Oncogenic phenotype assessment was done in 2D culture and two different 3D models; 1) Solid tumor model, in which cancer cell interaction with extracellular matrix and stromal cells, were represented in multiculture-spheroid system, 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid. Results: Dissimilar oncogenic phenotypes were seen among RP-R07, UOK-109 and UOK-146 in 2D and 3D culture. The presence of nuclear E-Cadherin was observed in all three cells model, with increasing level of nuclear expression in fusion partner dependent manner (P<0.05). Distinctive profiles of global phosphotyrosine and global phosphoserine were observed in cells model harboring different TFE3 fusions, despite common nuclear signal of TFE3. Nuclear expression of selected kinases and phosphokinases such as AKT, FAK, mTOR, VGEFR-2 were identified among TFE3 fusion cells with different level of expression (P<0.05). These results suggest distinct alteration of kinases nucleocytoplasmic shuttling regulation in tRCC cells (P<0.05), as compared to non TFE3 fusion cells. TFE3 ChIP-seq data in RP-R07 indicate novel DNA target involving metabolism and TGF-β signaling related genes. Conclusions: Our results indicate a distinct biology underlying tRCC oncogenesis in different TFE3 fusion models involving kinase reprograming and nucleocytoplasmic shuttling regulation. These differences also suggest the possibility of generating personalized treatments targeting specific TFE3 fusion genes associated with the spliceosome machinery. Citation Format: Nur P. Damayanti, Sreenivasulu Chintala, Ashley Orillion, Remi Adelaiye-Ogala, May F. Elbanna, Pete Hollenhorst, Roberto Pili. Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2017-4475

Abstract 3540: Novel targeted combinational therapies for melanoma

Abstract The BRAFV600E mutation is frequently observed in over 60% of melanomas. Development of the small molecule inhibitor PLX4032, which specifically targets the mutated BRAF protein, offers a novel therapeutic approach by effectively blocking the activation of key signaling molecules of the mitogen-activated protein kinase (MAPK) pathway, such as Mek and Erk, effectively leading to arrest in cell proliferation. Provided that MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesized that the inhibition of BRAFV600E can modulate the expression of MAAs. Since MAAs are essential in the development of a melanoma vaccine, due to their increased immunogenicity, altering their expression can be a useful immunotherapeutic approach to targeting melanoma. Using patient derived melanoma cell lines, which express various MAAs; our laboratory has developed a vaccinia virus based immunotherapeutic vaccine. These melanoma cell lines have been characterized by their status of BRAFV600E mutation and found to be either homozygous or heterozygous for the mutation. Inhibition of two key MAPK pathway molecules, namely Mek and Erk, denotes that the melanoma cell lines are responsive to PLX4032. Phenotypically, the cells’ invasive and migratory properties were also modulated upon treatment with PLX4032. Moreover, we observed that the expression of various MAAs was enhanced upon treatment with PLX4032. An amplification of gp100, MART-1 and tyrosinase expression on the RNA level as well as on the protein level was observed. Lastly, expression of MMAs was conserved in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. Based on our observations in that PLX4032 modulates MMA expression, further studies are warranted to determine the clinical effectiveness of combinational immunologic therapy using BRAFV600E inhibitors with our vaccinia virus melanoma cell line derived preparation. Citation Format: Robert Suriano, Neha Y. Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Novel targeted combinational therapies for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2015-3540

Abstract A30: Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma

Abstract As the BRAFV600E mutation is observed in over 60% of melanomas, the development of the small inhibitor, PLX4032, which specifically targets mutated BRAF, represents a novel therapeutic approach. This targeted inhibition of BRAF effectively blocks activation of key signaling molecules of the mitogen activated protein kinase (MAPK) pathway, such as Mek and Erk, thus halting cell proliferation. In addition, since MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesize that the inhibition of BRAFV600E can modulate the expression of MAAs thus serving as a viable target for immunotherapy of melanoma. Our lab has developed a vaccinia virus based immunotherapeutic vaccine using patient derived melanoma cell lines which express various MAAs. The melanoma cell lines have been characterized as far as the status of the BRAFV600E mutation and were found to be either homozygous or heterozygous for the mutation and are responsive to PLX4032, as evidenced by inhibition of two key MAPK pathway molecules, namely Mek and Erk. Phenotypically, the cells invasive and migratory properties were also modulated upon treatment with PLX4032. Interestingly was the observation that upon treatment with PLX4032, the expression of various MAAs was enhanced. Expression of gp100, MART-1, and tyrosinase increased both on the RNA level as well as on the protein level. The expression of MMAs were well maintained in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. We conclude that an effective multivalent immunotherapeutic vaccine requires the use of various MAAs and since PLX4032 upregulates expression of MAAs, further studies are warranted to determine the clinical effectiveness of combining BRAFV600E inhibitors with our melanoma cell line derived preparation. Citation Format: Robert Suriano, Neha Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A30.

Abstract 641: Targeted inhibition of BRAF signaling in melanoma enhances the activity of a multivalent immunotherapeutic vaccine

Abstract Development of a successful immunotherapeutic vaccine targeting melanoma requires the use of multiple melanoma associated antigens (MAA). Expression of several MAAs is regulated by the micropthalmia transcription factor (MITF), which in turn is regulated by members of the MAPK pathway, such as ERK. It is also known that 70% of melanomas express the genetic lesion BRAFV600E. Based on this knowledge, we hypothesize that the use of a specific BRAFV600E inhibitor would enhance MITF mediated MAA expression and thus serve as a viable target for immunotherapy. We have developed a vaccinia virus based melanoma vaccine using patient derived primary melanoma cells that are rich in MAAs such as gp100, tyrosinase, and TRP-1. Our results indicated that our cells were either homozygous or heterozygous for the mutated BRAF gene. In addition, our cells were responsive to treatment by PLX-4032, an FDA approved treatment for melanoma, as evidenced by inhibition of phosphorylation of MEK and ERK, two key players in the MAPK pathway. Phenotypically, the migratory and invasive properties of our cell lines were also altered by PLX-4032. Most interestingly was the observation that upon treatment with PLX-4032, the expression of various MAAs was altered. Specifically, the expression of gp100, MART-1, and tyrosinase increased both on the RNA level as well as on the protein level. Our results warrant further investigation into a combinatorial therapeutic approach to treat melanoma by which increasing the expression of various immunologically relevant MAAs, upon treatment with PLX-4032, followed by vaccination may lead to a more robust and targeted anti-melanoma immune response. Citation Format: Robert Suriano, Neha Tuli, Jan Geliebter, Raj K. Tiwari, Marc K. Wallack. Targeted inhibition of BRAF signaling in melanoma enhances the activity of a multivalent immunotherapeutic vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2014-641

Tumor and inflammation markers in melanoma using tissue microarrays

The importance of tumor immune response is ever more evident in melanoma carcinogenesis. One approach to explore the pathological mechanisms involved in such immune responses, and to analyze other tumor prognostic markers in melanoma, is to use tissue microarrays (TMAs). However, TMA technology remains to be adequately validated in this setting. Protein expression patterns in whole slides and TMA sections from 34 melanoma patients were compared for a number of inflammation and tumor cell markers using immunohistochemical stains against CD8 (lymphocytes), CD163 (macrophages), micropthalmia transcription factor, N-cadherin, melanoma cell-adhesion molecule, and c-kit protein (CD117). Using simplified versions of previously published grading systems, the agreement between TMA and whole slide sections ranged between 83 and 96%, and between 81 and 97% for inflammation and tumor cell markers, respectively. We conclude that TMA technology combined with simplified grading systems are appropriate for analyzing both inflammation and tumor cell markers in melanoma.

Canine malignant melanoma alpha-3 integrin binding peptides

There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 h. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma.

Cellular Neurothekeoma With Fascicular Growth Features Mimicking Cellular Dermatofibroma

Cellular neurothekeoma (CNT) is a benign cutaneous mesenchymal neoplasm. Most demonstrate a lobulated to micronodular architecture. Rarely, CNT demonstrates a predominantly fascicular growth pattern, without prominent sclerosis and thus can mimic cellular dermatofibroma (DF). Three CNT with a predominantly fascicular pattern were obtained. The clinicopathologic features and accompanying immunohistochemical stains were evaluated. All cases demonstrated a moderately cellular proliferation of epithelioid to spindle cells with pale to eosinophilic slightly granular cytoplasm, vesicular nuclei, and a single nucleolus arranged in a fascicular pattern with thick collagen bundles at the periphery (collagen trapping). One case had prominent epidermal hyperplasia. The neoplastic cells expressed NKI-C3, CD10, and micropthalmia transcription factor and lacked expression of factor XIIIa, S-100, epithelial membrane antigen, and CD34. Our cases showed an unusual pattern of CNT with a predominantly fascicular growth pattern, thickened collagen bundles at the periphery, and occasionally epidermal hyperplasia. The overlap with cellular DF is striking. The presence of plump to epithelioid cytomorphology with abundant cytoplasm, with focally prominent nucleoli; the presence of focal lobulated to micronodular growth pattern along with micropthalmia transcription factor positivity; and lack of factor XIIIa expression are helpful in distinguishing fascicular CNT from cellular DFs.

SOX10 immunostaining distinguishes desmoplastic melanoma from excision scar

Sry-related HMG-BOX gene 10 (SOX10), a nuclear transcription factor that plays an important role in schwannian and melanocytic cell differentiation, has recently been shown to be a useful marker in the diagnosis of melanocytic and schwannian tumors. Fibroblasts and histiocytes that could histopathologically mimic melanoma cells often express S100, which complicates the evaluation of melanoma excision specimens for residual tumor. Distinguishing melanoma cells from immature fibrocytes or histiocytes is made more challenging in desmoplastic melanoma excision specimens. We compared the utility of melanoma markers [SOX10, S100, HMB-45, Melan-A and micropthalmia transcription factor (MiTF)] in 3 invasive, 9 desmoplastic and 14 intraepidermal melanomas. We also evaluated 18 excision scars. The staining intensity for all the cellular components in melanoma and scar specimens was scored. SOX10 strongly highlighted all in situ, invasive and desmoplastic melanomas. In contrast, MiTF expression was weak to absent in desmoplastic melanomas. In scars, S100 highlighted background spindled fibrocytes and histiocytes with greater intensity than SOX10. MiTF highlighted multi-nucleated histiocytes, while SOX10 did not. Our results showed that SOX10 was strongly expressed by desmoplastic melanoma. Furthermore, SOX10 was less likely than S100 and MiTF to be expressed by background fibrocytes and histiocytes within scars.

Idiosyncrasies of Scalp Melanoma

Examine the accuracy of sentinel lymph node biopsy (SNB) in scalp melanoma (SM), patterns of nodal metastases, patient outcomes, and the utility of immunohistochemistry (IHC) in SNB evaluation. Retrospective. There were 22 patients, 4 females and 18 males. Sentinel lymph nodes (SLN) were localized via preoperative lymphoscintigraphy, intraoperative gamma probe, and Lymphazurin injection. SLNs were stained with hematoxylin-eosin, S-100, HMB-45, Melan-A, micropthalmia transcription factor, and tyrosinase. SLNs were grouped into cervical (levels 1-5) and extracervical (parotid, suboccipital, retroauricular) regions. There were 13 posterior and 9 anterior SMs. The first SNB were mapped to the extracervical regions in 77% of posterior and 78% of anterior lesions. SLN number ranged from 1 to 5. Ten patients had positive SLNs (PSLN). Forty percent of the PSLN group had SLNs mapped in both cervical and extracervical sites. Six underwent completion lymphadenectomy, with no additional positive nodes identified. No significant difference between PSLN and negative sentinel node (NSLN) patients was seen when compared by SLN number, Breslow's thickness, tumor ulceration, and clinical outcomes. Mean follow-up was 35 months. One patient died of disease. One isolated regional recurrence occurred. Sixty percent of PSLN and 92% of NSLN patients were recurrence free at last follow-up. One distant metastasis occurred in the NSLN group, and one local, one regional, and two patients with distant metastases were in the PSLN group at the time of last follow-up. Additional IHC did not detect other metastases in the NSLN group. SM is aggressive, as demonstrated by the high rate of SLN metastases, and there were no significant histopathologic factors in the primary tumor that predicted the presence of SLN metastases. SNB was accurate. The majority of first SLNs were localized in extracervical basins.

The Regulation of Cathepsin K Gene Expression

Cathepsin K is essential for normal bone resorption. Osteoclasts synthesize and secrete cathepsin Kinto the extracellular compartment at the attachment site between osteoclasts and the bone surface, wherein the organic matrix is subsequently degraded by cathepsin K. RANKL, NFAT, Mitf, and various components of AP-1 enhance osteoclast formation and bone resorption, whereas IFN-gamma, calcitonin, estradiol, and calcium inhibit it. These agents appear to act correspondingly to alter cathepsin K mRNA and protein expression in order to stimulate and suppress the osteoclast's resorbing potential. RANKL signaling via the calcineurin-calcium-NFAT signaling cascade plays a significant role in the regulation of cathepsin K expression. Activation via p38 and the micropthalmia transcription factor also enhances cathepsin K expression. Future studies will be needed to elucidate the relative roles of various signaling pathways at different stages of osteoclast formation and activation and to determine whether genetically disrupting these pathways can modulate bone resorption with or without impeding other osteoclast functions.

Mechanisms of Skin Tanning in Different Racial/Ethnic Groups in Response to Ultraviolet Radiation

Ultraviolet radiation stimulates pigmentation in human skin, but the mechanism(s) whereby this increase in melanin production (commonly known as tanning) occurs is not well understood. Few studies have examined the molecular consequences of UV on human skin of various racial backgrounds in situ. We investigated the effects of UV on human skin of various races before and at different times after a single 1 minimal erythemal dose UV exposure. We measured the distribution of DNA damage that results, as well as the melanin content/distribution and the expression of various melanocyte-specific genes. The density of melanocytes at the epidermal:dermal junction in different types of human skin are remarkably similar and do not change significantly within 1 wk after UV exposure. The expression of melanocyte-specific proteins (including TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (tyrosinase-related protein 2), MART1 (melanoma antigens recognized by T-cells) gp100 (Pmel17/silver), and MITF (micropthalmia transcription factor)) increased from 0 to 7 d after UV exposure, but the melanin content of the skin increased only slightly. The most significant change, however, was a change in the distribution of melanin from the lower layer upwards to the middle layer of the skin, which was more dramatic in the darker skin. These results provide a basis for understanding the origin of different skin colors and responses to UV within different races.

Single UVB Overexposure Stimulates Melanocyte Proliferation in Murine Skin, in Contrast to Fractionated or UVA-1 Exposure

Overexposure to short- and long-wave ultraviolet radiations (UVB, UVA) may contribute to melanoma development through combined genotoxic and mitogenic effects in melanocytes. This study compares the impact of UVA-1 versus UVB, and single versus fractionated exposures on melanocyte proliferation in hairless SKH-2 mice. A single erythemal dose was compared with an equal dose fractionated over 8 d, and dose-dependency was studied. Proliferation (Ki-67 positive-sign) in melanocytes (melanoma antigen recognized by T-cells-1 positive or micropthalmia transcription factor positive) was ascertained in double-labeled skin sections. Single erythemal UVB exposures caused a delayed, dose-dependent increase of melanocyte proliferation. The highest, 17-fold, increase (from 0.05% to 0.8% of melanocytes) occurred 4 d after UVB exposure, without any detectable effect on overall melanocyte numbers. Correspondingly, DNA repair-deficient xeroderma pigmentosum A (Xpa) mice proved exquisitely sensitive to melanocyte proliferation induction by UVB exposure. No discernable effects were measured from fractionated suberythemal UVB exposures, or from any UVA-1 exposure regimen. Hence, melanocyte proliferation appears to be most efficiently induced by a single UVB overexposure. Moreover, the ineffectiveness of UVA-1 radiation and the enhanced sensitivity of Xpa mice point at pyrimidine dimers as causative DNA lesions. Consequently, murine nevi and melanoma are expected to be most effectively induced by intermittent UVB overexposures.

Hematopoietic Expression of Hoxb4 Is Regulated in Normal and Leukemic Stem Cells through Transcriptional Activation of the Hoxb4 Promoter by Upstream Stimulating Factor (Usf)-1 and Usf-2

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.