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First published online 30 April 2003
doi: 10.1242/jcs.00472

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PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression

Dipartimento di Biologia Cellulare e dello Sviluppo, Università, Viale delle Scienze, 90128 Palermo, Italy

View larger version (33K): [in a new window]   Fig. 1. (A) DD-PCR of cDNA preparations from control (C) and PTHrP-treated (T) 8701-BC cells. The arrow points to a band of approximately 180 bp, selectively displayed in lane T (detail of a 6% sequencing PAGel, silver staining). (B) PCR amplification of the 168 bp product of hsbp1 cDNA in control (C) and PTHrP-treated cells (T). A band of the expected size can be observed in both lanes. Agarose gel 2%, ethidium bromide stain. M=100 bp size marker. (C) SM-PCR for hsbp1. Representative plot of normalized data versus cycle numbers fit with an exponential curve for control (s) and PTHrP-treated (•) 8701-BC cells.

View larger version (39K): [in a new window]   Fig. 2. (A) Panel of PCR analyses showing the presence of amplification products for hsp10, hsp60, hsp90, hsp90ß, mthsp75, hsf1 and hsf2 in both preparations from control (C) and PTHrP-treated (T) 8701-BC cells. No signal was found for hsp27, hsp70, hsc70 and grp78 (ethidium bromide stain). (B) Histogram showing the increase in the expression level of hsp10, hsp90, hsp90ß, hsf1 and hsf2, as quantitated by SM-PCR assay.

View larger version (70K): [in a new window]   Fig. 3. (Top) Western immunodetection of hsf1, hsf2 and hsp90 in lysates of control (A) and PTHrP-treated (B-E) 8701-BC cells grown in unsupplemented medium (B) or supplemented with 600 nM hsbp1-asODN (C), 1 µg geldanamycin/ml (D) or 100 µM quercetin (E). (Bottom) Coomassie Blue stain of SDS-PAGel after protein transfer to check loading.

View larger version (48K): [in a new window]   Fig. 4. PCR amplification of uPa (A,B) and MMP-1 (C,D) cDNA fragments in samples from control (A,C) and PTHrP-treated 8701-BC cells (B,D). M=100 bp-size marker (agarose gel 2%, ethidium bromide stain).

View larger version (33K): [in a new window]   Fig. 5. (Left) Histogram showing the decrease of uPa expression level in PTHrP-treated cells following incubation with 600 nM hsbp1-asODN or 1 µg geldanamycin/ml. (Right) Ethidium bromide-stained agarose gels of cDNA of preparations from scrambled-asODN-treated (A), hsbp1-asODN-treated (B), control untreated- (C) and geldanamycin-treated 8701-BC cells (D) cultured in the presence of 1 nM PTHrP [67-86]-amide, amplified in multiplex by primers for MMP-1 and ß-actin.

View larger version (12K): [in a new window]   Fig. 6. SM-PCR for uPa (A) and MMP-1 (B). Representative plots of normalized data versus cycle numbers fit with an exponential curve for untreated- (s) and quercetin-treated (•) 8701-BC cells.

View larger version (29K): [in a new window]   Fig. 7. Chemoinvasion response of scrambled-asODN-treated- (A), hsbp1-asODN-treated (B), control untreated- (C) and geldanamycin-treated (D) 8701-BC cells to PTHrP [67-86]-amide. E and F report the invasive activity of 8701-BC cells seeded in plain and quercetin-containing RPMI 1640 medium, respectively. The histogram shows a decrease in the number of migrated cells down to approximately 20% (B) and 34% (D) and an increase of up to approximately 3.6-fold (F) of controls.