Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: Serial sections of grade I SCC (A–C) and normal skin (H–J) were immunostained with antibodies against CCHCR1, Ki67, and EGFR. Higher magnifications of A–C are also shown (D–F, respectively). CCHCR1 staining (G) in an adjacent section to EGFR staining (F). CCHCR1 protein (A) is expressed in proliferative cancer cells at the invasive front of dermal cancer cell islands of an SCC in association with the hyperproliferation marker Ki67 (B) and EGFR (C). The Ki67 positive cells (E) express CCHCR1 (D). EGFR staining (F) associates with CCHCR1 staining (G) in adjacent sections. Normal skin samples express CCHCR1 (H) and EGFR (J) in basal KCs, while Ki67 expression (I) is more sparse. Arrows point at illustrative positions. Scale bars: (A–C) 50 µm; (D–G) 12.5 µm; (H–J) 25 µm.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Staining, Marker, Expressing

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: Serial sections of grade III (A, B) and grade II (C, D) SCC were stained with the indicated antibodies. Higher magnification of another grade II SCC (E, F). In grade III SCC (A, B), expression of CCHCR1 is heterogeneous: many, but not all, cancer cells express both CCHCR1 (A) and Ki67 (B). The insets show lower magnification of the same region, asterisk points out a cancer cell with negative CCHCR1 and Ki67 expression. In grade II SCC (C, D), infiltrative outgrowth is CCHCR1 positive (C), and the CCHCR1 positive cancer nests are also cyclin-D1 positive (D). Adjacent sections of grade II SCC (E, F) show association of CCHCR1 and cyclin-D1 positive cells. Arrows indicate corresponding areas. Scale bars: (A,B,E,F) 12.5 µm; (C,D, insets) 50 µm.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: Serial sections of BCC (A, B) were stained with antibodies against CCHCR1 and EGFR. Higher magnifications of A and B are also shown (C, D, respectively). CCHCR1 protein is expressed in a granular pattern in the cytoplasm of the palisading cancer cells of a nodular BCC (A, C). CCHCR1 expression (C) co-localizes with EGFR staining (D). CCHCR1 and EGFR are also expressed in basal KCs (A, B). Arrows show corresponding positions. Scale bars: (A,B) 50 µm; (C,D) 25 µm.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: CCHCR1 transfected HaCaT cells (A–D) stained with antibodies against CCHCR1 (A) and EGFR (B). Overlay of A and B is shown in (C), and DAPI staining showing nuclei in (D). Cells expressing CCHCR1 (green) were also positive for EGFR (red) but the two proteins did not co-localize (C). CCHCR1 positive (arrow) and negative (arrow head) cells exhibit similar EGFR expression suggesting that CCHCR1 overexpression does not affect EGFR expression (B).

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Transfection, Staining, Expressing, Over Expression

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: Serial sections of KA (A, B, E, F) were stained with the antibodies against CCHCR1 and EGFR (A, B) or against CCHCR1 and Ki67 (E, F). Higher magnifications of A and B are shown (C, D, respectively). The pushing border of a KA is CCHCR1 positive (A) co-localizing with EGFR (B). Another sample is shown (E) with a prominent lymphocyte infiltrate and cytoplasmic CCHCR1 expression of the pushing border cells. More scarce Ki67 expression (F) is defined to the same areas but not necessarily to the same cells. Arrows indicate corresponding positions. Scale bars: (A, B, E, F) 50 µm; (C, D) 12.5 µm.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: Sections of Bowen's disease (A–D) immunostained with antibodies against CCHCR1 and EGFR as indicated. Serial sections of psoriasis (E, F) were stained with antibodies for CCHCR1 and Ki67. AK samples (G, H) were stained with CCHCR1 antibodies. In Bowen's disease (A, B), spongiosis, inflammatory infiltrate, and capillary proliferation are associated with CCHCR1 expression while areas lacking inflammation and spongiosis express less CCHCR1 (C). EGFR expression (D) in basal and suprabasal KCs of dermal papillae associates with CCHCR1 expression (B). Also in psoriasis (E), CCHCR1 is expressed basally or suprabasally (arrows) overlying the dermal papillae (dp) while rete ridges (rr) projecting into the dermis are almost negative for CCHCR1. In contrast, Ki67 expression (F) localizes to regions that are almost negative for CCHCR1 cells (arrowheads). AK samples (G) express CCHCR1 basally and suprabasally in association with KC heterogeneity, spongiosis or inflammation but adjacent areas with less inflammation or spongiosis (H) stain only faintly. Scale bars: (A–D) 50 µm; (E, F) 25 µm; (G, H) 12.5 µm.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: A) Ki67, EGFR, cyclin-D1 and CCHCR1 gene expression profile of five normal epidermal KC and eight cutaneous SCC cell lines (heatmap). Signal values of the probe sets were compared to the mean signal values of each probe set in KCs. The colouring is based on the log2 values of the change in the signal values. The up-regulated genes are shown in red and down-regulated genes are shown in green. B) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of one CCHCR1 and one Ki67 probe set in the HG-U133 Plus 3.0 array. Pearson's correlation coefficient R = 0.88. C) CCHCR1 and D) Ki67 mRNA expression levels in the normal KCs and cutaneous SCC cell lines as measured by qRT-PCR (TaqMan). Expression levels of CCHCR1 and Ki67 in the normal KCs and SCC cell lines were analyzed by qRT-PCR and corrected for the β-actin mRNA levels in the same samples. E) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of CCHCR1 and Ki67 mRNA levels. Pearson's correlation coefficient R = 0.46.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: CCHCR1 (A), Ki67 (B), and EGFR (C) mRNA expression levels (TaqMan) in HaCaT cells after treatment with the tumor promoters OA and menadione. CCHCR1 (D), Ki67 (E) and EGFR (F) mRNA expression in HaCaT, A5, II4, RT3, A431, and FaDu cells. The invasive cells II4 and FaDu and metastatic RT3 cells expressed less CCHCR1 (D) and Ki67 (E) than HaCaT and A5 cells. A431 cells (F) express EGFR mRNA clearly more than other cell lines. The ras-transformed clones A5, II4, and RT3 and FaDu cells express EGFR less than HaCaT cells. Quantitative RT-PCR results are shown relative to mRNA levels from corresponding control cells (assigned the value 1). Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Expressing, Transformation Assay, Clone Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

doi: 10.1371/journal.pone.0006030

Figure Lengend Snippet: A) Relative expression of CCHCR1 mRNA (as measured by TaqMan) at different time points (from 24 h to 8 d) after releasing the cells from serum starvation and high density culturing. Expression of CCHCR1 did not differ between confluent (control) cells and quiescent (starved) cells. Control cells expressed CCHCR1 2.5-fold more than proliferative HaCaT cells (24–48 h, 3–4 d). As confluency was reattained (8 d), the expression of CCHCR1 increased to the level of the control cells. B) Correlation between relative CCHCR1 and Ki67 expression levels. When CCHCR1 mRNA expression levels were compared to those of Ki67, a negative correlation was seen, confirming the proliferative status of the HaCaT cells. C) EGFR mRNA expression correlated with CCHCR1 mRNA expression. D) Correlation between CCHCR1 and Ki67 expression in the experiment with lower cell density. The negative correlation of CCHCR1 expression with Ki67 expression was even more profound as relative CCHCR1 mRNA decreased near to zero in the two control cells. E) Correlation between CCHCR1 and EGFR expression in the experiment with lower cell density. Here again, CCHCR1 expression correlated with EGFR expression. TaqMan PCR results are shown relative to mRNA levels from corresponding control cells assigned the value 1. Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Immunostainings of tissue sections were performed using the avidin-biotin-peroxidase complex method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, for CCHCR1 and cyclin-D1; or StreptABComplex/HRP Duet (Mouse/Rabbit), DAKO, A/S Glostrup, Denmark, no. K0492 for EGFR and Ki67).

Techniques: Expressing