Overexpression of Sp1 leads to p53‐dependent apoptosis in cancer cells

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro‐apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP‐Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub‐G1 fraction, caspase‐3 cleavage, and annexin‐V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53‐dependent apoptotic pathways in the wild‐type p53 cells but not in the p53‐mutated or p53‐deleted cell lines when these cells were infected with adeno‐GFP‐Sp1 virus. In addition, A549 (p53^+/+) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53^?/?) cell viability was significantly inhibited by adeno‐GFP‐Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression‐induced apoptotic cell death in transforming cells. © 2009 UICC     

In vitro evaluation of flavopiridol, a novel cell cycle inhibitor, in bladder cancer

Purpose: To determine the in vitro effects of flavopiridol on bladder cancer cell lines, immortalized urothelial cell lines, and normal urothelial cells well characterized for defects in p53, pRb, and p16. Methods: Growth inhibition was assessed via an MTT assay and apoptosis via DAPI nuclear staining. Cell cycle analysis was performed via propidium iodide staining and fluorescent activated cell sorting (FACS). Multidrug-resistant cells were generated by continuous exposure to doxorubicin. Results: Growth inhibition was not correlated with inactivation of p53, pRb, or p16. All cells experienced G2/M arrest within 24 h of flavopiridol exposure. Modest apoptosis was observed but required 72 h of continuous drug exposure to become evident. There was no obvious synergistic or antagonistic toxicity when flavopiridol was combined with radiotherapy or cisplatin dosed at the IC₅₀ despite the observation that radiotherapy and flavopiridol led to more profound G2/M arrest than either agent alone. Doxorubicin-resistant cells, demonstrated to overexpress the MDR1 multidrug-resistance protein were equally as sensitive to flavopiridol as the parental cells. Conclusions: Flavopiridol is a novel cell cycle inhibitor that may be a useful agent in bladder cancers with tumor suppressor gene alterations and/or multidrug resistance. Received: 7 July 1998 / Accepted: 28 October 1998     

Insulin lispro: in-vivo potency determination by intravenous administration in conscious rabbits.

Insulin lispro is a monomeric analogue of human insulin, produced by genetic engineering, and has been reported to have a more rapid absorption following subcutaneous injection than insulin. Since it has been shown to have a similar hypoglycaemic action to insulin in clinical studies and comparable properties in radioimmunoassay, the feasibility of using a bioassay which was designed originally for insulin, to measure insulin lispro potency was evaluated in this investigation. A random-dose bioassay protocol, in which insulin lispro and two insulin standards were administered intravenously in a random sequence, was used and validated in nine conscious healthy rabbits. The decline in blood-glucose levels, following the intravenous injection of a dose of insulin or its lispro analogue, was monitored by a continuous glucose monitoring system. A glucose response curve was generated, from which various pharmacodynamic parameters were determined. Compared with the insulin standards, the potencies of insulin lispro determined from nadir, basal glucose normalized nadir, glycaemic reduction and ABGC (area of the blood-glucose response curve under baseline) were observed to have mean (95% confidence limits) values of 97.0 (69.5-124.6)%, 106.3 (72.4-140.2)%, 949 (51.8-138.0)% and 102.4 (76.3-128.5)%, respectively. In addition, the coefficients of variation for correspondent parameters were 36.9, 41.5, 59.1 and 33.2%, respectively. The results indicated that the hypoglycaemic potency calculated from the ABGC values was the most accurate (102.4%) with the least coefficient of variation (33.2%). In conclusion, the potency of insulin lispro can be determined accurately from the ABGC values measured by the random-dose bioassay used.     

High serum level of matrix metalloproteinase-1 and its rapid surge after intervention in patients with significant carotid atherosclerosis.

High tissue matrix metalloproteinase (MMP) activity has been reported to be associated with atherosclerosis and plaque rupture. The aim of this study was to elucidate the diagnostic value of serum MMP-1 in carotid stenosis and its dynamic change after stenting. We measured high-sensitivity C-reactive protein (hs-CRP) and MMP-1 in 37 patients with carotid stenosis (>or= 50%) and 84 controls. In 30 patients who underwent stenting, MMP-1 and hs-CRP were assessed immediately after stenting. We found that patients with carotid stenosis exhibited significantly higher MMP-1 compared with controls, but there was no difference in hs-CRP. Moreover, MMP-1 was elevated immediately after stenting. In multivariate analyses, MMP-1 was negatively correlated with statin and angiotensin converting enzyme inhibitor/angiotensin-II receptor blocker use in controls. In conclusion, higher levels and rapid surge after stenting in patients with carotid stenosis indicate that MMP-1 is an important composition of plaques, and suggest its potential role in the assessment of plaque burden and stability of carotid stenosis.     

C ASE R EPORT: Dramatic response to lamivudine therapy following corticosteroid priming in chronic hepatitis B

A 21 year-old male patient with chronic hepatitis B was treated with lamivudine 150 mg daily after withdrawal of a short course of oral prednisolone (30 mg daily for 3 weeks, 15 mg daily for 1 week). Serum hepatitis B virus (HBV)-DNA increased during prednisolone pretherapy and serum alanine aminotransferase (ALT) was increasing after withdrawal of prednisolone. Clearance of HBV-DNA with hepatitis B e antigen seroconversion and ALT normalization occurred within 2 months after starting lamivudine therapy. If this dramatic response to lamivudine therapy after corticosteroid priming is confirmed by further studies, the regimens used in this particular case might become a powerful therapeutic tool for chronic HBV infection.