1993;128:273C277. does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5+ NK cells in the low-metastatic murine melanoma model investigated. INTRODUCTION Heat shock proteins (Hsps) are a group of physiologically essential highly conserved proteins that are induced by heat shock as well as by other environmental and pathophysiological stresses. They play an essential role in protein folding, assembly, and transport, and are considered to act as molecular chaperones. Furthermore, Hsps play a role in the regulation of cell growth and differentiation (Hightower 1991; Morimoto et al 1994). There is evidence that these molecules are also involved in immune reactions resulting in auto- or tumor immunity (Melcher et al 1998; Van der Zee et al 1998; Schild et al 1999; Wells and Malkovsky 2000). Hsp25 is the murine homolog of human Hsp27 and belongs to the family of small Hsps (sHsps). This group consists of diverse intracytoplasmic proteins with molecular weights of 15C30 kDa and a variety of functions, including roles in signal transduction, growth arrest, and differentiation (Ciocca et al 1993; Kindas-Mgge and Trautinger 1994; Huot et al 1998). We have previously reported that overexpression of Hsp27 by DNA transfer in a human melanoma cell line (A375) as well as in a human epidermal squamous carcinoma cell line (A431) leads to retardation of cell growth in vitro and a delay in tumor development in athymic BALB/c-nu+/nu+ mice (Kindas-Mgge et al 1996). Based on these findings the question arose whether the delayed tumor development observed was caused solely by the reduced proliferation rate of is under transcriptional control of the retroviral long-terminal repeats promoter. This plasmid was named pHsp25. A corresponding antisense construct (pHsp25-AS) was used as a control. Transfection and Canertinib (CI-1033) selection of target cells Subconfluent cell cultures of K1735-Cl23 and K1735-M2 were transfected with 20 g of the plasmid of interest using the calcium phosphate precipitation procedure (100-mm culture dish, Mammalian Transfection Kit; Stratagene, La Jolla, CA, USA). Cells were selected for neoresistance by Geneticin, a neomycin analog (0.5 mg/mL G418; Sigma, Munich, Germany). Single-cell colonies were isolated, cultivated in 60-mm culture dishes, and tested for overexpression of Hsp25 by Western blot analysis (polyclonal antibody to Hsp25; Stressgen, Victoria, BC, Canada), as previously described (Kindas-Mgge et al 1996). Stably transfected clones with according to Western blot analysis a strong overexpression of Hsp25 (K1735-Cl23: Cl23/6, Cl23/7, and Cl23/8; K1735-M2: M2/1, M2/3,and M2/5) were used as target cells in cytotoxicity assays. Antisense-transfectants (Cl23/6, Cl23/7, Cl23/8) and controls (Cl23/AS2, Cl23/AS3) even at higher E:T ratios. (a)C(c) Each datapoint represents the percentage of specific lysis of at least 3 independent experiments (each carried out in triplicate). Error bars indicate standard error of the mean (SEM) In order to determine which effector population mediated the effect described previously, DX-5+ NK cells were positively selected using an immunomagnetic method. FACS analysis revealed that the positively selected DX-5+ NK cells exhibited a purity of 90C95?%. Canertinib (CI-1033) The NK-depleted fraction showed a frequency of DX-5+ NK cells that was below 2?%. DX-5Cenriched Canertinib (CI-1033) and DX-5Cdepleted lymphocyte populations were incubated with recombinant murine IL-2 for 72 hours, and were consecutively used as effector cells. Cytotoxicity tests with these highly purified IL-2Cstimulated DX-5+ NK cells resulted in clearly enhanced susceptibility to lysis of K1735-Cl23 clones overexpressing Hsp25, compared with antisense-transfectants and controls even at higher E:T ratios. (a)C(c) Each datapoint represents percentage of specific lysis of at Canertinib (CI-1033) least 3 independent experiments (each carried out in triplicate). Error bars indicate standard error of the mean (SEM) Likewise, cytotoxicity Canertinib (CI-1033) tests with IL-2Cstimulated DX-5+ NK cells as effectors revealed no relevant difference in susceptibility to SIRT3 NK-mediated lysis between K1735-M2 cells overexpressing Hsp25 and antisense control cells (Fig 4b). Hsp25-overexpressing clones as well as the antisense control did not show susceptibility to lysis by IL-2Cstimulated NK-depleted effectors (Fig 4c). Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic potential FACS analysis was performed in order to investigate whether surface localization of Hsp25 would influence target cell susceptibility to killing. All clones showed surface display of Hsp25. No difference was detectable with regard to cell-surface expression of Hsp25 between clones overexpressing Hsp25 and antisense-(Cl23/AS2, Cl23/AS3) express Hsp25 in equal amounts on their cell surface. (b) Histograms of K1735-M2 cells. No difference with regard to cell-surface display of Hsp25 between clones overexpressing Hsp25 (M2/1, M2/3, M2/5) and control cells (M2/AS) is detectable. (a,b) Analysis was repeated 3 times; a representative set of results is shown Hsp25 expression does not influence cell-surface display of MHC class I We used FACS analysis to investigate whether Hsp25 overexpression would alter the MHC class I expression on the cell.