Elise S. Bales, Cheryl Dietrich, Debdutta Bandyopadhyay, Denise J

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High Levels of Expression of p27KIP1 and Cyclin E in Invasive Primary Malignant Melanomas Elise S. Bales, Cheryl Dietrich, Debdutta Bandyopadhyay, Denise J. Schwahn, Weidong Xu, Vladimir Didenko, Paula Leiss, Nicole Conrad, Olivia Pereira-Smith, Ida Orengo, Estela E. Medrano Journal of Investigative Dermatology Volume 113, Issue 6, Pages 1039-1046 (December 1999) DOI: 10.1046/j.1523-1747.1999.00812.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of p27KIP1, p21Waf-1/SDI-1, cyclin D1, cyclin E, and pRB in primary invasive melanoma tumors. (A, B) Expression of p27KIP1 and p21Waf-1/SDI-1 in tumor sections from patient 5. (C) Cyclin D1 expression in a tumor section from patient 8(2) (scale bars: 25 μM). (D) Cyclin E expression in a tumor section from patient 11 (scale bars: 25 μM). (E, F) Expression of p27KIP1 and pRB in sections from patient 20 (scale bars: 25 μM). Journal of Investigative Dermatology 1999 113, 1039-1046DOI: (10.1046/j.1523-1747.1999.00812.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 High levels of p27KIP1 do not favor apoptosis in the primary melanoma cells. (A) Multiple apoptotic thymus cells (positive control) from rats treated for 24 h with 6 mg per kg dexamethasone. Green fluorescence-TUNEL staining visualizing characteristic DNA condensation in the nuclear membrane (arrowheads); Blue-DAPI fluorescence staining showing cellular DNA content. (B) No indications of apoptosis are seen in melanoma cells localized in the reticular dermis in the subcutis. (C) High levels of p27KIP1 expression in the same tumor. Apoptosis was determined using the Apoptag Kit as described by the manufacturer. Identical results were obtained using the ligase method described by Didenko & Hornsby 1998 (data not shown). Scale bars: 25 μM. Journal of Investigative Dermatology 1999 113, 1039-1046DOI: (10.1046/j.1523-1747.1999.00812.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 p27KIP1 and p21Waf-1/SDI-1 expression in the human skin and skin appendixes. (A) p27KIP1 expression in isolated cells in the epidermal–dermal junction (arrows), stratum spinosum, isolated fibroblasts, endothelial cells and lymphocytes; (B) Ki-67 labeling; (C) p21Waf-1/SDI-1 expression in the epidermis and sweat ducts; (D) p21Waf-1/SDI-1 expression in the pilosebaceous unit: note the presence of strong p21Waf-1/SDI-1 staining in cells differentiating to form the hair shaft and some positive nuclei in sebocytes. Scale bars: 25 μM. Journal of Investigative Dermatology 1999 113, 1039-1046DOI: (10.1046/j.1523-1747.1999.00812.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CDK2-p27KIP1 complexes and CDK2 kinase activity in normal melanocytes, nevus melanocytes, and primary and metastatic melanomas. Five hundred micrograms of total cell extracts were immunoprecipitated with an anti-CDK2 antibody as described in Materials and Methods. The immunoprecipitates were used to determine CDK2-associated histone H1 activity (500 μg) and to determine associations of CDK2 with p27KIP1 and cyclin E (200 μg). Lane 1, normal human melanocytes (NHM) in proliferating medium (Medrano et al. 1994); lane 2, NHM in differentiating medium containing cholera toxin (Medrano et al. 1994); lane 3, primary invasive melanoma cells (PM) (to be described elsewhere) in proliferating medium; lane 4, metastatic melanoma cells IIB-Mel-J (MM) (Guerra et al. 1989); lane 5, human nevus melanocytes (NM) in proliferating medium. Journal of Investigative Dermatology 1999 113, 1039-1046DOI: (10.1046/j.1523-1747.1999.00812.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 High levels of cyclin E but not cyclin D1 expression in primary and metastatic melanoma cells. Total cell extracts from proliferating (P) and senescent (S) normal human melanocytes isolated from light skin (lanes 1 and 2) and dark skin (lanes 3 and 4) skin; primary invasive melanoma cells (lane 5) and IIB-Mel-J metastatic melanoma tumor cells (lane 6) were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted, and probed with anti-cyclin E, anti-cyclin D1, anti-CDK2, and anti-p27 antibodies (see Materials and Methods). All samples contained 30 μg of protein per lane. Journal of Investigative Dermatology 1999 113, 1039-1046DOI: (10.1046/j.1523-1747.1999.00812.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions