The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P ¼ 0.005), CD133 (P ¼ 0.003), and nestin (P ¼ 0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P ¼ 0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P ¼ 0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.
Overexpression of HER-2/Neu occurs in about 25-30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed postphosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself. Methods:Immunohistochemistry for Her2 and Pin1 were performed on two hundred twentythree human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence.Results: Sixty-four samples (28.7%) stained positive for Her2 (IHC 3+), and 54% (122/223) of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5%) were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2 degradation, which could be prevented by treatments with the proteasome inhibitor ALLnL. Conclusion:Pin1 is a novel regulator of erbB2 that modulates the ubiquitin-mediated degradation of erbB2. The overexpression of Pin1 in a majority of Her2-overexpressing breast cancer may contribute to maintain erbB2 levels. Pin1 inhibition alone and in conjunction with mTOR inhibition suppresses the growth of Her2+ breast cancer cells.
Stem cells of the hair follicle are located in the bulge region of the follicular isthmus, in a region that specifically expresses keratin 15 (Lyle et al, 1998;Liu et al, 2003). Because stem cells can be localized in tissue samples, these cells are being increasingly studied to further elucidate hair biology. Some methods of study use fluorescent antibodies, the presence of which is noted by exciting the reacted tissue with specific and limited wavelengths of light. We report the phenomenon of autofluorescence by cells in the human hair follicle, strikingly localized to the bulge region, incidentally observed upon reviewing negative controls in fluorescent antibody studies. The presence of this autofluorescence must be considered when analyzing immunofluorescent studies to avoid potential misinterpretation. In addition, the findings add insights into the biochemical nature of stem cells.Follicles were prepared from skin obtained from facelift procedures with Institutional Review Board approval and in compliance with the declaration of Helsinki guidelines. Skin samples from two patients were rapidly frozen in OTC and then 5 mm frozen sections were cut. Additional samples were used to obtain plucked follicles by the following technique. The skin was placed in Dulbecco's modified Eagle medium (DMEM) supplemented with penicillin and streptomycin. The tissue was cut into pieces approximately 1 cm wide and 1-3 cm long. Fascia and deep fat below the follicular bulbs were removed. These trimmed fragments were then placed in DMEM containing 4 mg per mL of Dispase (Sigma, St. Louis, Missouri), and digested either overnight at 41C or 1-3 h at 371C. Following digestion, the tissue was washed once in sterile phosphate-buffered saline (PBS), and then placed into fresh DMEM on ice. Hair follicles were plucked under a dissecting microscope and segregated according to the stage of the hair cycle. Plucked whole follicles and frozen sections were then examined under a Zeiss Axiophot fluorescence microscope using Zeiss DAPI (Carl Zeiss, Inc., Thornwood, New York), fluorescein isothiocyanate (FITC), and rhodamine filters. The DAPI #02 filter excites at 365 nm and emits at 420 nm (blue); the FITC #09 filter excites between 450 and 490 nm and emits at 515 nm (green); and the rhodamine #15 filter excites at 546 nm and emits at 590 nm (red). Follicles studied under the FITC and rhodamine filters were coverslipped with PBS or Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, California), which helps preserve fluorescence.
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