Nuclear iASPP may facilitate prostate cancer progression.

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPP(Δ8/Δ8) mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.

SUMO-modified nuclear cyclin D1 bypasses Ras-induced senescence.

Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19(Arf)/p21(waf1/cip1)-independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated.

Limited value of serum holo-transcobalamin II measurements in the differential diagnosis of macrocytosis.

AIM: To study the value of serum holo-transcobalamin II (holo-TCII) measurements in the differential diagnosis of macrocytosis. METHODS: Holo-TCII concentrations were measured in serum samples from 50 healthy non-vegetarian subjects and 30 patients with macrocytosis, using a technique based on the adsorption of holo-TCII with amorphous, precipitated silica. Deoxyuridine (dU) suppression tests were performed on the bone marrow cells of all the patients. Haematological diagnoses were made using standard criteria. RESULTS: The causes of macrocytosis were cobalamin (Cbl) deficiency due to pernicious anaemia or following partial gastrectomy (10 patients), dietary folate deficiency with/without Cb1 deficiency (four patients), chronic alcoholism (four patients), myelodysplastic syndrome (five patients), treatment with methotrexate or azathioprine (three patients), and congenital dyserythropoietic anaemia (CDA) (four patients). Undetectable or low holo-TCII concentrations were found in all patients with Cb1 deficiency and in some or all patients from each of the other diagnostic categories. There was also no correlation between the dU suppressed value and the holo-TCII concentration: all 15 patients with high dU suppressed values and nine of 15 with normal dU suppressed values, including four patients with CDA, had low holo-TCII concentrations. CONCLUSIONS: Measurements of serum holo-TCII concentrations by the silica adsorption method are not of value in the differential diagnosis of macrocytosis. The finding of low serum holo-TCII concentrations in patients with macrocytosis due to causes other than Cb1 deficiency may result not only from a state of negative Cb1 balance but also from other factors, such as increased utilisation of holo-TCII as a consequence of erythroid hyperplasia.

Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity.

Research into the pathogenesis of acetaminophen (paracetamol)-induced hepatotoxicity has concentrated on the generation of toxic metabolites by the hepatocytes. It has, however, recently been shown that human macrophages cultured with acetaminophen secrete increased quantities of tumour necrosis factor (TNF). This study examines whether macrophages have a direct role in acetaminophen toxicity, using a mouse model in which it is possible to eliminate more that 99 per cent of hepatic macrophages by previously injecting liposomes containing dichloromethylene disphosphonate (DMDP). Acetaminophen-induced liver damage was assessed biochemically and histologically. It was shown that the liver damage occurring 0.5, 1, and 2 h after an intraperitoneal injection of acetaminophen was significantly less in mice previously injected with liposomes containing DMDP than in previously untreated mice, or mice previously injected with empty liposomes. By 4 h there was no difference between the groups. We conclude that macrophages play an early and probably a direct role in mediating the liver damage due to acetaminophen. This is consistent with the role that macrophages have been shown to play in the pathogenesis of alcohol-induced liver damage.

The effect of Kupffer cell elimination on ethanol-induced liver damage in mice.

C57BL/10 mice develop inflammatory and necrotic changes in the liver, as well as raised serum ALT activities, after 9 days of exposure to ethanol vapour. If mice were injected twice with liposomes containing dichloromethylene diphosphonate (DMDP), with an interval of 5 days between the injections, there was complete elimination of Kupffer cells (hepatic macrophages) for a 9-day period starting 1 day after the first injection. The inflammatory and necrotic changes were significantly reduced in mice injected with liposomes containing DMDP as compared to uninjected mice or mice injected with empty liposomes; serum ALT activities were also significantly reduced. No significant difference was seen in serum tumour necrosis factor-alpha levels between the different groups. Kupffer cells therefore play a significant role in the development of the liver damage resulting from exposure to ethanol. Acetaldehyde production by Kupffer cells is one way in which these cells can damage hepatocytes and further work needs to be done to investigate this and other mechanisms.

A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment.

The binding or precipitation of DNA onto gold or tungsten microcarriers represents one of the most crucial steps for gene transfer via the particle bombardment process. We have developed a simple and rapid method to monitor DNA precipitation onto microcarriers before delivery to intact cells or tissues. Binding of DNA constructs to different microcarriers was evaluated with relative fluorescence values using a dedicated fluorometer. Significantly greater precipitation was detected using gold vs. tungsten microcarriers. Addition of glycerol resulted in a 46% increase in precipitation. A 42% difference in precipitation was observed using two different brands of polyproplyene tubes. Fluorescence values dropped 10-50% 3 hr after initial precipitation. Fluorescence values were correlated with the number of transient GUS transformants of rice (Oryza sativa, L.) cells. Precipitation with PEG gave higher fluorescent values and GUS transformants than a similar method without PEG. Results from these experiments indicate that fluorescence measurements are an effective and rapid method to monitor DNA precipitation for particle bombardment experiments.