Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins-MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.

Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer.

Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

Hyperoxia fully protects mitochondria of explanted livers.

Liver ischemia-reperfusion injury is still an open problem in many clinical circumstances, including surgery and transplantation. This study investigates how mitochondrial structure, mass and oxidative phosphorylation change and may be preserved during a brief period of ischemia followed by a long period of reperfusion, an experimental model that mimics the condition to which a liver is exposed during transplantation. Livers were explanted from rats and exposed for 24 h to three different oxygen availability conditions at 4 °C. Mitochondrial mass, respiration, oxidative phosphorylation (OXPHOS), and levels of OXPHOS complexes were all significantly altered in livers stored under the currently used preservation condition of normoxia. Remarkably, liver perfusion with hyperoxic solutions fully preserved mitochondrial morphology and function, suggesting that perfusion of the graft with hyperoxic solution should be considered in human transplantation.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells.

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive disease, nevertheless exhibiting a high response rate to chemotherapy. Since the retinoblastoma protein (pRb) loss confers a high sensitivity to chemotherapy regimens, we evaluated the prevalence of pRb loss in TNBCs and its relevance on the clinical outcome of patients treated with adjuvant chemotherapy. PATIENTS AND METHODS: pRb status was prospectively evaluated by immunocytochemistry in 518 consecutive patients with complete receptor information. The predictive value of pRb status in TNBCs was determined according to the adjuvant therapeutic treatments. RESULTS: Fifty-three tumors were identified as TNBCs. The prevalence of pRb loss was significantly higher in TNBCs than in the other cancer subtypes. All patients with TNBCs lacking pRb and treated with systemic chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil) were disease free at a medium follow-up time of 109 months, whereas the clinical outcome of those expressing pRb was significantly poorer (P = 0.008). Analysis of disease-free survival including the established anatomo-clinical prognostic parameters indicated pRb loss as the only significant predictive factor. CONCLUSIONS: pRb loss is much more frequent in TNBCs than in the other breast cancer subtypes. Patients with TNBCs lacking pRb had a very favorable clinical outcome if treated with conventional adjuvant chemotherapy.

Endoscopic appearance of GERD: putative role of cell proliferation.

BACKGROUND: Erosive esophagitis is a frequent endoscopic feature in patients with gastro-oesophageal reflux disease. However, most of patients with heartburn/regurgitation have a non-erosive reflux disease. The reason for this heterogeneous impact of gastro-oesophageal reflux disease on oesophageal mucosa is unknown to date. AIM: To evaluate the cell proliferation status of oesophageal epithelium in both healthy normal subjects and patients with gastro-oesophageal reflux disease with or without erosions. MATERIALS AND METHODS: All the subjects underwent endoscopy and biopsies were taken at 5 cm from the squamo-columnar junction. Specimens were analysed both at histology and at transmission electron microscopy. Cell proliferation was evaluated by MIB1 immunostaining. Of the 85 subjects were studied, 10 were healthy controls with normal pH-testing and macroscopical, histological and ultrastructural patterns; 37 were patients with erosive esophagitis, and 38 patients with non-erosive reflux disease. RESULTS: At histology, of the 37 patients affected by erosive esophagitis, 30 had normal mucosa and 7 showed mild oesophagitis. One patient with non-erosive reflux disease showed signs of oesophagitis at histology. At TEM, all patients with gastro-oesophageal reflux disease had ultrastructural patterns of damage i.e. dilations of intercellular spaces (DIS), and all controls had a normal ultrastructural pattern. The mean (+/-SD) MIB1-LI values of normal subjects and non-erosive reflux disease and erosive oesophagitis patients were 62.2% (+/-9.1), 29.7% (+/-7.2) and 16.2% (+/-5.2), respectively; there were significant differences among the three groups (p<0.001). CONCLUSIONS: Oesophageal mucosa of patients with reflux symptoms presents a decrease in MIB1 immunostaining of 50% and 25% in non-erosive reflux disease and erosive esophagitis patients with respect to normal subjects.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient human osteosarcoma cell lines.

OBJECTIVES: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. MATERIAL AND METHODS: RB- and p53-proficient U2OS cells and the RB- and p53-deficient SAOS-2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. RESULTS: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G(1)-S and G(2)-M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS-2 cells. No effect on cell cycle progression after actinomycin D-induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS-2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS-2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS-2 cells after the long-term actinomycin D treatment, whereas no modification was observed in U2OS cells. CONCLUSIONS: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB- and p53-deficient cells. A daily-repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer.

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.

Relationship between the RB1 mRNA level and the expression of phosphorylated RB protein in human breast cancers: their relevance in cell proliferation activity and patient clinical outcome.

The aim of the present study was to ascertain the relationship between the level of RB1 mRNA and the expression of phosphorylated RB protein and the relevance of these two parameters in cancer cell proliferation and clinical outcome in human breast cancer. Sixty-eight primary human breast cancers were considered. The amount of RB1 mRNA was evaluated by quantitative RT-PCR analysis. The level of RB phosphorylation was immunohistochemical defined by measuring the phosphorylated (pp) RB labelling index (LI). Cell proliferation rate was measured by calculating the Ki67 LI. No relation was found between the RB1 mRNA level and the ppRB LI (p=0.565). Both RB1 mRNA value and ppRB LI were related (in an inverse and direct manner, respectively) to Ki67 LI. RB1 mRNA expression was more strictly associated with KI67 LI (p=0.001) than the ppRB LI (p=0.013). Regarding the patient clinical outcome, the separately considered RB parameters did not reach the prognostic significance. However, patients with low RB1 mRNA quantity and patients with high ppRB LI, taken together, had a significantly shorter disease free and overall survival than the group comprehending patients with high RB1 mRNA value and low ppRB LI, and this despite the low number of patients considered. Our results demonstrated that the ppRB LI was independent of the RB1 mRNA level; that both RB parameters are related to the cell proliferation rate and, if collectively considered, have a high informative value on breast tumour prognosis.

Prognostic relevance of a novel semiquantitative classification of Bcl2 immunohistochemical expression in human infiltrating ductal carcinomas of the breast.

BACKGROUND: Bcl2 is an important prognostic parameter in human breast cancer. However, the evaluation of Bcl2 expression by immunohistochemistry is carried out using arbitrary scoring criteria. In the present study, we evaluated the clinical relevance of a novel, semiquantitative classification of the Bcl2 immunostaining based on both the distribution and the intensity of the staining reaction. PATIENTS AND METHODS: The proposed classification was first validated in 69 breast cancer specimens by comparing the Bcl2 immunostaining with the Bcl2 messenger RNA (mRNA) levels evaluated by real-time RT-PCR. Since a highly significant association was found between protein and mRNA for Bcl2, the immunohistochemical scoring system was applied to 442 patients with infiltrating ductal carcinomas of the breast with long-term follow-up (median observation time 106 months). RESULTS: In the entire series, the Bcl2 variable was an independent predictor of clinical outcome, and its prognostic independence was maintained when lymph node-negative and -positive patients were considered separately. In this regard, of particular interest was the observation of a subgroup of node-negative breast cancer patients with a negative Bcl2 immunostaining, who had a very high probability of relapse or death (respectively about five and seven times greater than patients with a positive Bcl2 immunostaining). Moreover, the Bcl2 variable retained prognostic significance also in subgroups of patients treated with either adjuvant endocrine therapy or chemotherapy. CONCLUSIONS: Our results demonstrated that in breast cancer, Bcl2 protein expression parallels its mRNA level, and it has a highly significant and independent prognostic relevance.

Dyskerin expression influences the level of ribosomal RNA pseudo-uridylation and telomerase RNA component in human breast cancer.

Dyskerin is a nucleolar protein, altered in dyskeratosis congenita, which carries out two separate functions, both fundamental for proliferating cells. One function is the pseudo-uridylation of ribosomal RNA (rRNA) molecules, necessary for their processing, and the other is the stabilization of the telomerase RNA component, necessary for telomerase activity. A significant feature of dyskeratosis congenita is an increased susceptibility to cancer; so far, however, no data have been reported on dyskerin changes in human tumours. Therefore, in this study, the distribution of dyskerin in a large series of human tumours from the lung, breast, and colon, as well as from B-cell lymphomas, was analysed by immunohistochemistry. Dyskerin proved never to be lost or delocalized outside the nucleolus. A quantitative analysis of dyskerin mRNA expression was then performed in 70 breast carcinomas together with the evaluation of telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin mRNA levels were highly variable and directly associated with both telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin gene silencing in the MCF-7 human breast carcinoma cell line reduced telomerase activity and rRNA pseudo-uridylation. Significantly, patients with low dyskerin expression were characterized by a better clinical outcome than those with a high dyskerin level. These data indicate that dyskerin is not lost in human cancers and that the levels of its expression and function are associated with tumour progression.

A statistical model predicting high hepatocyte proliferation index and the risk of developing hepatocellular carcinoma in patients with hepatitis C virus-related cirrhosis.

BACKGROUND: Incidence of hepatocellular carcinoma in hepatitis C virus-related cirrhosis is 4% per year. Although cost-effective, current screening could be improved. AIM: To develop a statistical model including non-invasive parameters able to identify patients at high risk of developing hepatocellular carcinoma. METHODS: One hundred and fifty-eight patients (73F:85M) with compensated chronic hepatitis C virus liver disease underwent evaluation, including argyrophilic nucleolar organizer regions proliferation index, and were followed up for 56.18 +/- 1.44 months. RESULTS: Fifty-six patients had chronic hepatitis without cirrhosis and low argyrophilic nucleolar organizer regions proliferation index (< or =25%), 65 had hepatitis C virus-related cirrhosis and low argyrophilic nucleolar organizer regions proliferation index and 37 had hepatitis C virus-related cirrhosis and high argyrophilic nucleolar organizer regions proliferation index (>25%). Groups were similar for gender and viral genotype distribution. None of the patients with chronic hepatitis without cirrhosis developed hepatocellular carcinoma, compared with 6.1% of low argyrophilic nucleolar organizer regions proliferation index and 30.6% of high argyrophilic nucleolar organizer regions proliferation index (P = 0.002). By multivariable logistic regression analysis, the following parameters were independently associated with hepatocellular carcinoma development and used for the development of the statistical model: platelets (OR 0.98), gamma-globulins (OR 0.111), alanine aminotransferase/aspartate aminotransferase ratio (OR 0.07), serum ferritin (OR 1.0) and ultrasonographic pattern (coarse OR 2.9, coarse nodular OR 10.12). The statistical model properly allocated 95.9% of patients with low argyrophilic nucleolar organizer regions proliferation index and 72.2% of patients with high argyrophilic nucleolar organizer regions proliferation index. CONCLUSIONS: The model, to be validated in large prospective studies, may help tailoring screening according to the risk of hepatocellular carcinoma development.

The prognostic value of the AgNOR parameter in human breast cancer depends on the pRb and p53 status.

BACKGROUND: The amount of argyrophilic nucleolar organiser regions (AgNORs) represents a cell kinetics parameter used in tumour pathology for prognostic purposes. AgNOR expression is directly related to the rate of ribosome biogenesis, which has been recently shown to be controlled also by the tumour suppressor proteins pRb and p53. AIMS: To ascertain the relative prognostic value of AgNORs and of pRb and p53 expression in breast carcinoma. METHODS: This study was carried out on 335 human primary breast carcinomas with a median follow up time of 108 months. AgNORs were measured by morphometric analysis on sections that had been selectively silver stained; expression of p53 and phosphorylated and non-phosphorylated pRb forms was assessed by immunohistochemistry. RESULTS: Patients were divided into groups with low (249) and high (86) AgNOR values; with normal (267) and mutated p53 (68); and with normal (256) and hyperphosphorylated or deleted pRb (79). Univariate analysis of disease free survival showed that AgNORs and the status of pRb and p53 were significantly related to the patients' clinical outcome. However, among the four groups characterised by different pRb and p53 status, the AgNOR parameter was not capable of distinguishing subgroups of patients with different clinical outcomes. CONCLUSIONS: These findings indicate that the prognostic value of the AgNOR parameter depends on the status of the tumour suppressor proteins pRb and p53, and it cannot be ascribed to the relation between AgNORs and the cell proliferation rate.

Thymidylate synthase protein expression and activity are related to the cell proliferation rate in human cancer cell lines.

AIMS: To ascertain whether the expression and enzyme activity of thymidylate synthase (TS) are related to the rapidity of cell proliferation in human cancer cell lines. METHODS: Ten asynchronously growing human cancer cell lines of different origin were used, characterised by various doubling times. TS expression was evaluated by western blot analysis using the TS 106 monoclonal antibody. TS activity was determined by the enzyme catalytic assay. The quantitative variation of TS in different phases of the cell cycle was investigated using two parameter flow cytometry for the TS protein and DNA analysis. The number of proliferating cells was evaluated by Ki67 immunostaining. RESULTS: TS expression and activity were significantly related to each other (r = 0.765; p = 0.01) and to the cell doubling time (r = -0.899; p < 0.001 and r = -0.919; p < 0.001, respectively). Ki67 immunolabelling showed no association between the number of cycling cells and TS protein expression and activity. Two parameter flow cytometry indicated that differences of TS expression in the cell lines were not related to the cell cycle phases or to the proportion of S phase cells. CONCLUSIONS: These results show that the expression and activity of the TS protein in asynchronously growing cancer cells are significantly related to the cell doubling time; the faster the cell proliferation, the greater the expression and activity of TS. These findings could explain why TS values are of prognostic value per se and why tumours with high TS expression benefit more from chemotherapy.

[Silver-stained Nucleolar Organizer Regions (AgNOR)]. / Stato dell'arte sulle AgNOR.

The present paper discusses the relevance of silver-stained Nucleolar Organizer Regions (AgNOR) to tumour pathology. First, the structural and functional aspects of AgNORs and the biological meaning of their quantitative variations have been reviewed to clearly define which indications can be obtained by the use of AgNOR. In continuously proliferating cells, AgNOR indicates the extent of ribosomal biogenesis, which is strictly related to the rapidity of cell proliferation. Therefore, AgNOR can be considered to represent a marker of cell proliferation rate and, in tumour pathology, should be used only for prognostic purposes. The predictive value of AgNOR, the only parameter which indicates the cell growth rate in situ in routinely processed cyto-histological samples, is strengthened by the combination with markers of cell growth kinetics (e.g. Ki67/MIB1).

Coupling of 5-fluoro 2'-deoxyuridine to lactosaminated poly-l-lysine: an approach to a regional, non-invasive chemotherapy of liver micrometastases.

Nucleoside analogs conjugated with galactosyl-terminating peptides selectively enter liver cells and after intracellular release from the carrier partly exit into bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. The aim of the present experiments was to ascertain whether, in mice injected with non-toxic doses of a 5-fluoro 2'-deoxyuridine (FUdR) conjugate with lactosaminated poly-L-lysine (L-poly(LYS)), the drug was released by hepatic cells in high enough amounts to be pharmacologically active on neoplastic cells infiltrating the liver. We observed that L-poly(LYS)-FUdR inhibited the growth of hepatic metastases induced by intrasplenic administration of murine colon carcinoma C-26 cells. L-poly(LYS)-FUdR was not toxic for C-26 cells in vitro, was selectively taken up by mouse liver, and was stable in mouse blood, indicating that the effect on the metastases was due to FUdR (and/or its active metabolites) released in liver blood after the conjugate was taken up by the hepatic cells. These results suggest that L-poly(LYS)-FUdR might be useful in adjuvant chemotherapy of tumors giving liver metastases. The drug released from hepatic cells into liver blood following conjugate administration via the peripheral venous route might accomplish a locoregional, non-invasive treatment of micrometastases nourished by liver sinusoids.

Can large cell change and high proliferative activity predict hepatocellular carcinoma in patients with hereditary hemochromatosis?

OBJECTIVE: Patients with hereditary hemochromatosis are at high risk of developing hepatocellular carcinoma. This study was undertaken to define whether large cell change and nucleolar organizer regions proliferative index (marker of high proliferative activity) predict hepatocellular carcinoma development in hereditary hemochromatosis. METHODS: Histological staining for large cell change and high proliferative activity were done on baseline liver biopsies of 74 patients with hereditary hemochromatosis (52 with and 22 without cirrhosis), prospectively followed-up for 83 +/- 53 months (range, 1-230 months). RESULTS: Large cell change and high proliferative activity were found only in cirrhotic patients; 16 of 52 patients (31%) had either the large cell change or high proliferative activity. Large cell change was more frequent in patients with hepatitis B surface antigen than in those positive for hepatitis C virus (57% vs 14%, p = 0.04). Hepatocellular carcinoma developed in 7 of 16 (44%) and in 6 of 36 patients (16%) of the patients positive or negative for these morphological variables. The probability of developing hepatocellular carcinoma at 7 yr of follow-up was significantly higher in patients with large cell change or high proliferative activity than in those without. The length of follow-up from baseline histology to hepatocellular carcinoma occurrence was shorter in patients with large cell change or high proliferative activity than in those without these changes (46 +/- 36 and 109 +/- 34 months, p = 0.01). A multivariate analysis indicated that in patients with cirrhosis, large cell change or high proliferative activity (considered as a single variable), and age >55 yr were the only independent variables significantly associated with the risk of developing hepatocellular carcinoma, with a risk ratio of 4.8 (confidence interval 1.2-18.2) and 4.0 (confidence interval 1.1-15.6), respectively. CONCLUSIONS: In hereditary hemochromatosis, the presence of large cell change or high proliferative activity in patients older than 55 yr with cirrhosis should be considered a strong predictor of hepatocellular carcinoma development, especially if hepatitis B virus infection coexists.

p120 expression provides a reliable indication of the rapidity of cell duplication in cancer cells independently of tumour origin.

p120 is a nucleolar protein that has been immunocytochemically detected in rapidly proliferating cells of a variety of human malignancies. In the present study, the relationship between p120 expression and the rapidity of cell proliferation was evaluated in 48 human tumours of different origins. The cell proliferation rate of cancer cells was determined by quantitative analysis of AgNOR proteins. p120 immunostaining and AgNOR protein quantity were measured by image cytometry and a highly significant correlation was found between the two variables, as evaluated by linear regression analysis (r=0.98, p<0.0001). The relationship between p120 expression and the rapidity of cell duplication was also studied in vitro, in six human cancer cell lines derived from different tumour types, characterized by various doubling times (ranging from 20 to 77 h). p120 expression was determined on western blots using specific anti-p120 monoclonal antibodies. Densitometric analysis revealed a highly significant inverse correlation between the integrated optical density values of the chemoluminescence bands at 120 kD and the cell line doubling times (r=-0.93; p=0.007). The same result was obtained in situ by correlating p120 immunostaining of the cytological preparations obtained from the six cancer cell lines with the corresponding doubling time (r=-0.98, p<0.0001). These results indicate that in cancer cells, the quantitative expression of p120 is directly related to the rapidity of cell duplication, independently of the tumour origin.

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.

In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin-fixed and paraffin-embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=-0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=-0. 99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6+/-5.6 SD and 95. 5+/-2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions.

The AgNORs.

The structure and the function of interphase AgNORs and the importance of the "AgNOR" parameter in tumor pathology have been reviewed. Interphase AgNORs are structural-functional units of the nucleolus in which all the components necessary for ribosomal RNA synthesis are located. Two argyrophilic proteins involved in rRNA transcription and processing, nucleolin and nucleophosmin, are associated with interphase AgNORs and are responsible for their stainability with silver methods, thus allowing interphase AgNORs to be visulaized at light microscopic level, also in routine cyto-histopathological preparations. The number of interphase AgNORs is strictly related to rRNA transcriptional activity and, in continuously proliferating cells, to the rapidity of cell proliferation. Evaluation of the quantitative distribution of interphase AgNORs has been applied in tumor pathology both for diagnostic and prognostic purposes. The "AgNOR" parameter has been proved to represent a reliable tool for defining the clinical outcome of cancer disease, being an independent prognostic factor in many types of tumors.