PIGMENT CELL RES 14: 456–465. 2001 Copyright © Pigment Cell Res 2001 Printed in Ireland—all rights reser6ed ISSN 0893-5785 Original Research Article RelA, p50 and Inhibitor of kappa B alpha are Elevated in Human Metastatic Melanoma Cells and Respond Aberrantly to Ultraviolet Light B SUSAN E. McNULTY, NILOUFAR B. TOHIDIAN and FRANK L. MEYSKENS Jr. Department of Medicine and Chao Family Comprehensi6e Cancer Center, Uni6ersity of California Ir6ine Medical Center, 101 City Dri6e South, Orange, California 92868 *Address reprint requests to Prof. Frank L. Meyskins, Chao Family Comprehensi6e Cancer Center, Building 23, Rte 81, Uni6ersity of California Ir6ine Medical Center, 101 City Dri6e South, Orange, California 92868. E-mail: fl[email protected] Received 20 April 2001; in final form 15 August 2001 Metastatic melanomas are typically resistant to radiation and melanocytes. We also found that melanoma cells expressed chemotherapy. The underlying basis for this phenomenon may higher cytoplasmic levels of RelA, p105/p50 and the inhibitory result in part from defects in apoptotic pathways. Nuclear protein, inhibitor of kappa B alpha (IkBa) than melanocytes. factor kappa B (NFkB) has been shown to control apoptosis in To directly test whether RelA expression has an impact on many cell types and normally functions as an immediate stress melanoma cell survival, we used antisense RelA phosphoroth- response mechanism that is rigorously controlled by multiple ioate oligonucleotides and found that melanoma cell viability inhibitory complexes. We have previously shown that NFkB was significantly decreased compared with untreated or con- binding is elevated in metastatic melanoma cells relative to trol cultures. The constitutive activation of NFkBin normal melanocytes. In the current study, Western blot analy- metastatic melanoma cell cultures may, therefore, support an sis showed that, compared with normal melanocytes, inappropriate cell survival pathway that can be therapeutically melanoma cell lines have higher nuclear levels of the NFkB manipulated. subunits p50 (7-fold) and RelA (5–10-fold). In response to tumor necrosis factor-alpha (TNFa), both melanocytes and Key words: Melanocyte, Melanoma, NFkB, Antisense, TNF, melanoma cells showed increased nuclear p50 and RelA levels, UVB but levels in melanoma cells remained higher than in INTRODUCTION The incidence of melanoma is increasing at the second Nuclear factor kappa B (NFkB) activation has been highest rate of all human cancers, and metastatic disease is shown to have both pro- and anti-apoptotic functions in generally fatal. Metastatic melanomas are typically resistant various cell types (5, 6). There are five mammalian NFkB/ to chemotherapy (1), with response rates of 15–20% Rel family members, p50, p52, RelA, RelB, and cRel, that achieved with dacarbazine, the best single agent, and re- share a highly conserved 300-amino acid Rel homology ported response rates ranging from 15 to 43% with combi- domain containing dimerization, nuclear localization, and nation dacarbazine, cisplatin, carmustine, and tamoxifen, DNA binding regions (5–8). These proteins can form homo- but with no survival benefit (2–4). It has been suggested and heterodimers, which bind DNA at NFkB sequences that the underlying resistance of metastatic melanomas to found in the promoters of a variety of genes and specific radiation and chemotherapy may be due at least in part to dimer pairs elicit differential induction of these genes (5, 8, over-expression of apoptosis inhibitory proteins (1). 9). Several studies have suggested that NFkB transcription Abbre6iations –NFkB, nuclear factor kappa B; IkB, inhibitor of kappa B; UVB, ultraviolet light B 456 Pigment Cell Res. 14, 2001 factors are associated with the genesis of a variety of cell lines. NFkB functions as an immediate stress response cancers, including colon, breast, and ovarian (10, 11). The mechanism, which is normally rigorously controlled by mul- activation of NFkB by tumor necrosis factor (TNF), ioniz- tiple inhibitory complexes. However, in human metastatic ing radiation, or daunorubicin was found to protect against melanoma cells, we have found constitutive NFkB activa- apoptosis (12). Inhibition of NFkB nuclear translocation tion that is not inhibited by the normal regulatory enhanced apoptotic killing by these reagents (12–16) but mechanisms. not by apoptotic stimuli mediated by an NFkB-independent pathway (12). Studies in RelA-deficient mice showed that MATERIALS AND METHODS the presence of this NFkB subunit is required for protection from TNFa (13, 14), and that RelA controls inducible, but Cell Culture not basal, transcription in NFkB-regulated pathways (14). Two to five human neonatal foreskins were placed in 0.25% Apparently, TNFa transmits one signal that induces cell trypsin at 4°C overnight as has been previously described death and another, dependent on RelA, that protects (25, 26). The tissues were scraped to recover the against cell death (13). melanocytes, pooled and cultured in MCDB 153 (Sigma) Nuclear localization and NFkB activity are tightly con- medium containing 2% fetal calf serum, 0.3% bovine pitu- trolled by inhibitory proteins, including p105 and p100, itary extract (Clonetics Corp.), 10 ng/ml phorbol myristate- which are the precursors for p50 and p52, as well as 13-acetate, 2.0 mM calcium chloride, 5 mg/ml insulin and 0.1 members of the inhibitor of kappa B (IkB) family, IkBa, b mM 3-isobutyl-1-methyl-xanthine (Sigma) (MCDB+). Hu- and o (5, 8, 16). p105 and p100 inhibit nuclear translocation man metastatic melanoma cells (c83-2C, c81-46A, c81-61) of heterodimers containing the transcriptional transactivat- were cultured in F-10 (Fisher Scientific) medium containing ing subunits RelA, cRel, and RelB. p50 and p52 homod- 5% fetal calf serum and 5% newborn calf serum (F10+). imers and p50/p52 heterodimers are able to translocate to Melanocyte and melanoma cultures were used in experi- the nucleus, but lack the NFkB transactivation domain and ments between passages 4 and 8. To compare the effects of generally act as transcriptional repressors (8). IkBa inhibits different culture conditions, cells were cultured for 24 hr p50 heterodimers containing cRel, RelA, and RelB (5, 8, prior to harvest as noted in specific experiments. 17), while IkBb preferentially inhibits RelA homo- and heterodimers (5, 8, 17, 18) and IkBo is associated with cRel UVB Irradiation k and RelA, but not p50 (5, 19). NF B binds to the promoters Cell cultures were grown to subconfluence, medium was for p100/p52, p105/p50, and IkBa and transcriptionally aspirated, cells were washed twice in phosphate-buffered activates their expression (8). Induction of unprocessed saline (PBS), a sufficient amount to cover cells was applied p105, p100, and IkB proteins by NFkB functions as a and the cell cultures were exposed to UVB in a range of negative feedback mechanism, which leads to down-regula- doses that clinically produce suberythemal (25 mJ/cm2)to tion of NFkB by sequestration of transactivating het- severe sunburn effects (100 mJ/cm2). The cells were irradi- erodimers as inactive cytoplasmic complexes. Induction of ated using a Stratalinker 2400 (Stratagene), which produces IkBa also facilitates export of activated RelA complexes UVB wavelengths in the range of 280–320 nm with a peak from the nucleus, thereby terminating NFkB function (20, at 302 nm. 21). However, persistent activation of NFkB by inducers such RNA Isolation, cDNA Probes and Northern Blot Analysis as LPS and IL-1 leads to a loss in cellular IkBa and IkBb (17, 22, 23), but despite rapid resynthesis of IkBa,NFkB Cytoplasmic RNA was isolated by detergent lysis in RSB remains induced. During prolonged NFkB stimulation, (25). The nuclei were pelleted by centrifugation and the IkBb is synthesized as an unphosphorylated protein that supernatant was diluted in urea buffer, extracted in phenol forms a stable complex with NFkB in the cytosol (22), but chloroform, and precipitated with ethanol. RNA was size does not mask the nuclear localization signal or DNA fractionated by denaturing formaldehyde–agarose gel elec- binding domain (23). Unphosphorylated IkBb then com- trophoresis and transferred to nylon filters by Southern transfer (27). The cDNA probes were labeled with [a- petes with IkBa and facilitates NFkB translocation to the 32P]dCTP (NEN) using random nonamer priming (Pharma- nucleus (23) and is one mechanism whereby NFkB may cia Biotech). The p50 cDNA probe was a 1.5-kb EcoRI become constitutively activated. insert, p65 was a 0.95-kb EcoRI insert, IkBa was a 1.2-kb While the etiology of human malignant melanoma is still EcoRI insert (gifts of John Hiscott, McGill University) (28), incompletely understood, there is substantial epidemiologi- and an 18S rRNA probe was used (Ambion, 18S Decatem- cal evidence that ultraviolet exposure resulting in sunburn, plate). The filters were hybridized with the [a-32P]dCTP-la- particularly during adolescence (24), is an important risk beled cDNA probes at 42°C for 2 hr in Rapid-Hyb Buffer factor. We have previously shown that NFkB expression (Amersham) and washed following the manufacturer’s and binding is altered in metastatic melanoma cells com- recommendation. pared with their normal melanocyte counterparts (25). Be- cause multiple signals activate NFkB, including UV irradiation (8), we investigated the effects of ultraviolet light Western Blot Analysis B (UVB) on the expression and function of NFkB-related Normal melanocyte and metastatic melanoma cell lines were proteins in normal melanocyte and metastatic melanoma harvested at subconfluence, and cytoplasmic and nuclear Pigment Cell Res. 14, 2001 457 fractions were prepared by published methods (29). The RESULTS cytoplasmic and nuclear proteins were quantitated by a NFkB Binding is Elevated in Metastatic Melanoma Cells modified Lowry assay (BioRad DC Protein Assay); Relative to Normal Melanocyte Cultures aliquots separated by sodium dodecylsulfate (SDS)-poly- Independent pools of normal melanocytes and metastatic acrylamide gel electrophoresis (PAGE) were stained by melanoma cell strains were grown to subconfluence and Coomassie blue to verify quantitation or were transferred nuclear and cytoplasmic fractions were isolated.