Postoperative intra-abdominal infection is an independent prognostic factor of disease-free survival and disease-specific survival in patients with stage II colon cancer.

BACKGROUND: Recurrence occurs in up to 20% of patients with stage II colon cancer operated on for cure. Although postoperative intra-abdominal infection has been linked with an increased risk of recurrence, the association is controversial. The aim was to investigate the impact of postoperative intra-abdominal infection on disease-free survival and disease-specific survival in patients with stage II colon cancer. METHODS: Patients undergoing elective surgery for colon cancer stage II, between 2003 and 2014, were included. Patients with anastomotic leak or intra-abdominal abscess were included in the infection group. We used the Kaplan-Meier method to represent the distribution of survival and the Cox proportional hazards model to estimate the contribution of relevant clinicopathological factors with prognosis. RESULTS: Postoperative intra-abdominal infection was diagnosed in 37 of 363 (10.2%) patients. Perioperative blood transfusion was more frequent in patients with infection (p = 0.008). Overall 5-year disease-free survival rate was 85.1%. Disease-free survival at 5 years was lower in patients with postoperative intra-abdominal infection (52.8 vs 88.7%; p < 0.001), perineural invasion (p = 0.001), lymphovascular invasion (p = 0.001), pT4 (p = 0.013), and in patients with adjuvant chemotherapy (p = 0.013). Multivariate analysis showed that postoperative intra-abdominal infection (HR 4.275; p < 0.001), perineural invasion (HR 2.230; p = 0.007), and lymphovascular invasion (HR 2.052; p = 0.016) were all significant independent predictors of reduced disease-free survival. Regarding specific survival, independent significant prognostic factors were the number of lymph nodes, lymphovascular invasion, and postoperative intra-abdominal infection. CONCLUSION: In this series of patients with stage II colon cancer, postoperative intra-abdominal infection has an independent negative impact on disease-free survival and disease-specific survival.

Postoperative serum Vascular Endothelial Growth Factor is an independent prognostic factor of disease free survival and overall survival in patients with non metastatic colon cancer.

BACKGROUND: Angiogenesis is essential for tumor growth and formation of metastasis. VEGF is the most potent agiogenic citokine. The aim was to investigate the predictive value of postoperative VEGF serum concentration in patients undergoing surgery for colon cancer. METHODS: Consecutive patients with colon cancer undergoing surgery with curative intent were included. VEGF was measured in serum at 48 h and postoperative day 4. Cox proportional hazards model was used to estimate its contribution with prognosis. RESULTS: A total of 94 patients were included. On multivariate analysis VEGF on postoperative day 4 (HR: 1.05; p = 0.011) was independent prognostic factor of decreased DFS and OS. Five-year DFS (57.7% vs. 85%; p = 0.001) and OS (93% vs. 72%; p = 0.005) were significantly lower in patients with postoperative serum VEGF greater than 370 pg/dl. CONCLUSION: Postoperative VEGF serum concentration was an independent predictor of recurrence. These results must be verified in a prospective independent testing cohort.

Peripheral blood leucocytes show differential expression of tumour progression-related genes in colorectal cancer patients who have a postoperative intra-abdominal infection: a prospective matched cohort study.

AIM: Anastomotic leak is associated with higher rates of recurrence after surgery for colorectal cancer. However, the mechanisms responsible are unknown. We hypothesized that the infection-induced inflammatory response may induce overexpression of tumour progression-related genes in immune cells. The aim was to investigate the effect of postoperative intra-abdominal infection on the gene expression patterns of peripheral blood leucocytes (PBL) after surgery for colorectal cancer. METHOD: Prospective matched cohort study. Patients undergoing surgery for colorectal cancer were included. Patients who had anastomotic leak or intra-abdominal abscess were included in the infection group (n = 23) and matched with patients without complications for the control group (n = 23). PBL were isolated from postoperative blood samples. Total RNA was extracted and hybridized to the Affymetrix Human Gene 1.0 ST microarray. RESULTS: Patients in the infection group displayed 162 upregulated genes and 146 downregulated genes with respect to the control group. Upregulated genes included examples coding for secreted cytokines involved in tumour growth and invasion (S100P, HGF, MMP8, MMP9, PDGFC, IL1R2). Infection also upregulated some proangiogenic genes (CEP55, TRPS1) and downregulated some inhibitors of angiogenesis (MME, ALOX15, CXCL10). Finally, some inhibitors (HP, ORM1, OLFM4, IRAK3) and activators (GNLY, PRF1, FGFBP2) of antitumour immunity were upregulated and downregulated, respectively, suggesting that the inflammatory environment caused by a postoperative infection favours immune evasion mechanisms of the tumour. CONCLUSION: Analysis of PBL shows differential expression of certain tumour progression-related genes in colorectal cancer patients who have a postoperative intra-abdominal infection, which in turn may promote the growth of residual cancer cells to become recurrent tumours.

Postoperative intra-abdominal infection and colorectal cancer recurrence: a prospective matched cohort study of inflammatory and angiogenic responses as mechanisms involved in this association.

BACKGROUND: Anastomotic leakage is associated with higher rates of recurrence after surgery for colorectal cancer. However, the mechanisms responsible are unknown. The aim was to investigate the inflammatory and angiogenic responses in patients undergoing surgery for colorectal cancer who had postoperative intra-abdominal infection, and to compare the results with patients without complications. METHODS: Consecutive patients undergoing surgery for colorectal cancer with curative intent were included. Patients who had an anastomotic leak or intra-abdominal abscess were included in the infection group and matched with patients who had an uncomplicated postoperative course. IL-6 and VEGF were measured in serum and peritoneal fluid. RESULTS: Serum concentration of IL-6 was higher in the infection group (n = 30) compared with the control group (n = 30) on day 4 (infection: 42.3 [27.6-1473.2] versus control: 0.6 [0.6-17.1] pg/ml; p = 0.008). IL-6 in peritoneal fluid was higher in the infection group at 48 h and day 4 (infection: 1000.2 [995.4-1574.0] versus control: 90.3 [35.2.6-106.1] pg/ml; p = 0.001). Serum VEGF was higher in the infection group on day 4 (infection: 1128.6 [427.3-10000.0] versus control: 438.3 [214.1-677.6] pg/ml; p = 0.001). Peritoneal VEGF concentration was higher in the infection group at 48 h and day 4 (infection: 10000.0 [2563.0-10000.0] versus control: 477.8 [313.5-814.4] pg/ml; p = 0.001). Two-year recurrence rate was higher in patients with infection (infection: 30% versus control: 4%; p = 0.001). CONCLUSIONS: Intra-abdominal infection increases IL-6 and VEGF after surgery for colorectal cancer. Amplification of inflammation and angiogenesis might be one of the mechanisms responsible for the higher recurrence rate observed in patients with anastomotic leakage or intra-abdominal abscess.

A phase II trial of the combination of gemcitabine, ifosfamide and cisplatin in the treatment of advanced non-small cell lung cancer.

OBJECTIVE: This phase II trial was designed to assess the efficacy and toxicity profile of the combination of gemcitabine, ifosfamide and cisplatin (GIP) in the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients included in the study were those with surgically unresectable or metastatic NSCLC, with bidimensionally measurable disease, a Karnofsky performance status > 60, and who had not received previous chemotherapy. Treatment consisted of 1000 mg/m(2) gemcitabine on days 1 and 8, 3 g/m(2) ifosfamide on day 1, and 50 mg/m(2) cisplatin on day 1, administered in 21-day cycles. A maximum of six cycles were administered. RESULTS: Between March 1996 and December 1997, 60 patients were included in the study (37 stage III and 23 stage IV), of which 59 were evaluated for response. An objective response was obtained in 43% of patients (3% complete and 40% partial responses), whereas 22% had stable disease. The median survival time for the whole group was 52 weeks (65 weeks in patients with stage III, and 35 weeks in stage IV). The most frequent toxicity was haematological, 56% of patients presented grade 3 or 4 myelotoxicity in one of the cycles, although only seven episodes of febrile neutropenia were recorded in the 255 cycles administered. CONCLUSIONS: The GIP regimen attains response rates similar to those obtained with the gemcitabine plus cisplatin combination used in advanced NSCLC, and had an acceptable toxicity profile.

E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein.

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.

Differential regulation of the retinoblastoma family of proteins during cell proliferation and differentiation.

In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.

The p130 pocket protein: keeping order at cell cycle exit/re-entrance transitions.

Pocket proteins, including the retinoblastoma susceptibility gene product (pRB) and the related proteins p107 and p130, function at cell cycle regulatory steps that link cyclin/CDK-integrated positive and negative growth signals with E2F transcription factor activity on genes required for cell cycle progression. Protein complex formation between pocket proteins and members of the E2F family of transcription factors determines whether E2F complexes act as transcriptional activators or repressors. Experimental work over the last few years indicates that individual pocket proteins interact with specific E2F members to regulate the transcription of certain genes under diverse cell growth conditions. Among these protein associations, p130-containing E2F complexes seem to be of particular importance in controlling gene transcription in quiescent and differentiating cells by repressing the transcription of a set of E2F-responsive genes. Once the cells are progressing through the G1 phase of the cell cycle, pocket protein-mediated regulation of E2F activity is assumed by pRB and p107. p130-mediated transcriptional regulation thus seems to prevent a gene expression program characteristic of dividing cells at the cell cycle exit and re-entrance transitions and in quiescent cells.

Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth.

The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell differentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and differentiation transitions.

pRB, p107 and p130 as transcriptional regulators: role in cell growth and differentiation.

The mammalian cell cycle engine, which is composed of cyclin/CDK holoenzymes, controls the progression throughout the cell cycle by regulating, at least in part, the transcription of two types of genes: genes whose protein products are required for DNA metabolism and genes whose protein products are involved in cell cycle control. Among the targets of cyclin/CDKs, there is a family of negative growth regulators collectively known as pocket proteins. This family of pocket proteins includes the product of the retinoblastoma tumor suppressor gene, pRB and the functionally and structurally related proteins p107 and p130. In this review, the mechanisms by which pocket proteins are thought to regulate cell growth and differentiation are discussed.

Phosphorylation site specificity of the CDC2-related kinase PITALRE.

PITALRE is a human protein kinase belonging to the cell division cycle 2 (CDC2) kinase family, and is the catalytic subunit of a multimeric complex that contains several cellular proteins. PITALRE complexes from several cell lines and tissues phosphorylate retinoblastoma protein and myelin basic protein (MBP). In the present work, we have found that MBP is phosphorylated by PITALRE complexes on both Ser and Thr residues. Two different antibodies raised to PITALRE purified virtually identical kinase activities, as analysed by MBP phosphopeptide mapping and phosphoamino acid analysis. We have identified the proline-directed residue Ser-162 of MBP as a major phosphorylation site for PITALRE. In addition, our results suggest that one of the two MBP proline-directed threonine residues, Thr-97, is also selectively phosphorylated by PITALRE. These data, together with analysis of different peptide substrates derived from sites on MBP that are phosphorylated by PITALRE, indicate that PITALRE is a Ser/Thr proline-directed kinase. In addition, our results show that PITALRE has a substrate site specificity distinguishable from those of the CDC2 and cyclin-dependent kinase 2 (CDK2).

The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo.

G1 cyclin/CDK-independent phosphorylation and accumulation of p130 during the transition from G1 to G0 lead to its association with E2F-4.

During the transition from G1 to G0, p130 undergoes a specific phosphorylation event-leading to p130-form 2- that is mediated by a kinase/s other than the known G1, S and G2/M cyclin/CDKs. Changes in the phosphorylation status of p130 during this transition are responsible, at least in part, for the concomitant formation of p130/E2F-4 complexes, which are characteristic of G0. These complexes remain abundant during early G1 upon restimulation, but not after mitosis, and are dissociated in mid G1 when p130 is abruptly hyperphosphorylated to form 3. Subsequently, p130 forms 1 and 2 are no longer detected during the remainder of the cell cycle. Consistently, phosphorylation to form 3 and dissociation from E2F-4 complexes is reproduced by a cyclin/CDK holoenzyme in vitro. TGF-beta-induced G1 arrest abrogates cyclin/CDK phosphorylation of p130 but not phosphorylation to form 2. The cell cycle-dependent phosphorylation pattern of p130 is thus shown to comprise two distinct steps that are catalyzed by different kinases. The differential regulation of p130 and pRB phosphorylation during the transition from G1 to G0 may explain the fact that p130 and E2F-4 are the major components of E2F complexes in quiescent cells. Moreover, the newly described phosphorylation of p130 at the transition from G1 to G0 defines a novel mechanism of cell cycle exit regulation.

Cell cycle-dependent phosphorylation of the retinoblastoma-related protein p130.

The retinoblastoma-related protein p130 is a putative negative regulator of cell proliferation in mammalian cells. In this study, p130 is shown to exist in multiple phosphorylated forms in human cells. In glioblastoma T98G cells synchronized by serum deprivation, specific phosphorylated forms of p130 are found at different times after serum re-stimulation. Two phosphorylated forms of p130 only found in serum-arrested T98G cells and in early G1 phase associate with the adenovirus oncoprotein E1A in vitro. One of these two forms corresponds to the in vivo E1A-associated p130 in 293 cells, which express endogenous E1A protein. Moreover, p130 undergoes an abrupt shift to a unique phosphorylated form in mid G1 which is the only p130 form found during the remaining phases of the cell cycle. This phosphorylated form possesses an associated histone H1 kinase activity that is most active in late S phase and G2/M. The cell cycle-dependent expression pattern of cyclins in T98G cells is compatible with cyclin D1/CDK complexes driving the shift to this phosphorylated p130 form in mid G1. These results suggest that the putative growth inhibitory function of p130 is regulated by phosphorylation of this protein. They also suggest that differential phosphorylation of p130 during the cell cycle plays distinct roles in the regulation of p130 function.

The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19.

A retinoblastoma-related human gene, referred to as RB2, has been cloned based on sequence homology of the E1A-binding domain of the retinoblastoma gene. Structural homology with the retinoblastoma gene suggests a possible function of RB2 as a tumor suppressor gene. In this study, we have mapped this gene to human chromosome 16q12.2 and rat chromosome 19, using fluorescence in situ hybridization and somatic hybrid cell analysis, respectively. Based on known syntenic relationships among human, rat and mouse, the data suggest that the mouse homolog resides on chromosome 8. Deletions of chromosome 16q have been found in several human neoplasias (including breast, ovarian, hepatic, and prostatic cancers) which is in support of an involvement of RB2 in human cancer as a tumor suppressor gene.

Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain.

The product of the retinoblastoma tumor suppressor gene (pRb) and p107 share a high degree of structural homology in the pocket region, which is thought to play a primary role in the function of these proteins. It is conceivable that there exists a larger family of cellular proteins containing this pocket region. In this communication, we report cloning of a new human cDNA encoding a polypeptide that shows a high level of identity with pRb and p107 and possesses a pocket region. We have named it pRb2. From the deduced amino acid sequence, pRb2 has a predicted molecular weight of approximately 120 kD and its in vitro translated product binds to the adenovirus E1A protein. Due to its size, pRb2 may correspond to p130, which has previously been shown by us to interact with the transforming region of E1A in in vivo studies. Interestingly, pRb2 fails to bind an E1A mutant in the transforming domain 2 suggesting that pRb2 is involved in the transforming capacity of E1A.

Ethinyl estradiol-induced cell proliferation in rat liver. Involvement of specific populations of hepatocytes.

Hepatocyte proliferation was analyzed in vivo during the time course of continuous administration to rats of the liver tumor promoter ethinyl estradiol (EE) at 10 p.p.m. in the diet. EE-induced acute liver hyperplasia was detected in male and female Sprague-Dawley rats as an increased mitotic index of hepatocytes after 2 days of treatment. 5'-Bromodeoxyuridine (BrdU) labeling showed that proliferating hepatocytes were randomly distributed throughout the hepatic lobule. Subsequently, and still during the first few days of continuous EE treatment, hepatocyte proliferation decreased to control levels, and a transient increase in the incidence of apoptosis in the liver was detected. Although consistent with the concept of liver growth regression after mitogen-induced hyperplasia, these results differ from others reported to date in that, in our experiments, the cessation of cell proliferation and the subsequent growth regression occurred without withdrawal of EE in our experiments. After returning to control levels, hepatocellular proliferation again increased between 3 and 6 months of chronic treatment and remained activated during the following months of continuous treatment, as seen by accumulative BrdU labeling. Proliferating hepatocytes were predominantly located in zone 2 of the hepatic lobule at this time, surrounding a periportal zone of vacuolated hepatocytes, which were also induced by the treatment. Moreover, hyperplasia of basophilic hepatocytes was also seen around some portal spaces. In another set of experiments, chronic EE-induced activation was characterized by flow cytometry on hepatocytes isolated from male Fischer rats. Ploidy analysis of hepatocyte cell suspensions showed that the normal polyploid pattern of hepatocytes was altered by EE, the proportion of diploid hepatocytes rising considerably. The results also showed that these diploid cells were the most susceptible hepatocyte population to EE-induced proliferation, as shown by a combination of BrdU labeling and cell sorting methods. In contrast to Sprague-Dawley rats, no vacuolated cells were found histologically in the livers of these animals and the proliferating hepatocytes were located adjacent to the portal areas. These results taken together support the existence of cell target populations in the liver responding to the effects of tumor promoters. The finding that a subpopulation of diploid hepatocytes was the liver cell class most susceptible to proliferation during chronic EE treatment may explain, at least in part, the behavior of EE as a tumor promoter in hepatocarcinogenesis.

Transforming growth factor-alpha expression in rat experimental hepatocarcinogenesis.

Growth factors in general and transforming growth factor-alpha in particular have been related to cell proliferation and cell differentiation. This study was designed to clarify the distribution pattern of TGF-alpha in chemically-induced hepatocarcinogenesis. Sprague-Dawley rats were subjected to different non-intensive or intensive carcinogenic treatments using diethylnitrosamine (DEN) as carcinogen and ethinyl estradiol (EE) as promoter. The livers were fixed in 2% paraformaldehyde, dehydrated in a series of ethanol solutions, embedded in paraffin and sectioned. In the preneoplastic lesions no TGF-alpha immunoreactive cells were identified, but in some hepatic tumours cell immunostained with TGF-alpha antibody were observed. These results suggest that the cells capable of expressing TGF-alpha constitutively may be involved in neoplastic development in vivo.

Distribution of fibronectin and fibronectin-binding proteins, AGp110 and integrin alpha 5 beta 1, during chemically induced hepatocarcinogenesis in adult rats.

We have used single- and double-label immunocytochemistry to examine the distribution of AGp110, integrin alpha 5 beta 1 and fibronectin in adult rat liver during carcinogenesis induced by aflatoxin B1 or diethylnitrosamine. In normal liver fibronectin and the fibronectin integrin receptor alpha 5 beta 1 are localized on all three domains of the parenchymal cell surface: sinusoidal, lateral and canalicular. In contrast, AGp110, a non-integrin monomeric glycoprotein with fibronectin receptor properties, is confined to the bile canalicular (apical) plasma membrane of hepatocytes. Hepatocarcinogenesis induced by aflatoxin B1 causes altered cell foci to form in the parenchyma, followed by enlargement of these foci to form pre-neoplastic nodules and finally hepatocellular carcinomas of either poorly differentiated, trabecular or adenocarcinoma morphology. Expression of AGp110 decreased to a minimal level, at first selectively in altered cell foci, from the 9th week of treatment, and then indiscriminately in poorly differentiated carcinomas. The same lesions that were deficient in AGp110 also displayed a reduced level of fibronectin and alpha 5 beta 1, although the observed change in AGp110 demarcated altered foci and poorly differentiated tumour lesions more sharply, since expression of alpha 5 beta 1 and fibronectin, though substantially reduced, was still faintly apparent on the cell surface. Small acinar structures, observed in late hyperplastic nodules and in trabecular carcinomas, exhibited even, pericellular staining of fibronectin and alpha 5 beta 1, including prominent staining of the lumen area, whereas staining of AGp110 appeared to be confined to the lumen. In larger ducts of overt adenocarcinomas, fibronectin and alpha 5 beta 1 were distributed along the basal surface of the epithelium and AGp110 on the apical domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell proliferation and tumour promotion by ethinyl estradiol in rat hepatocarcinogenesis.

A two-stage model of hepatocarcinogenesis is used to study the effect of exposure time to ethinyl estradiol (EE) on promotion of preneoplastic lesions in rat liver induced by diethylnitrosamine (DEN). Young male and female Sprague-Dawley rats initiated by a single dose of DEN (100 mg/kg) were subjected to different times of EE administration incorporated into the diet at 10 p.p.m. (0.5 mg/kg x day). Animals were killed 1 year after initiation. Whereas macroscopic tumours were rarely seen in animals with short exposure (3 or 4 months) or in only-initiated controls, all the animals under a long period of administration (8 months) showed macroscopic tumours. Morphometric studies on glutathione-S-transferase (GST) positive preneoplastic lesions revealed an increase in the mean size of foci and nodules corresponding to 8 months of treatment, whereas no changes were observed between animals with short exposure and only-initiated controls. No differences were seen in the incidence of these lesions between any of the protocols. In addition to an acute hyperplastic effect on non-initiated liver described earlier, our preliminary results suggest cytotoxicity and an enhancement of the liver cell turnover after several months of continuous EE administration. These results taken together suggest that promotion of hepatocarcinogenesis by EE largely depends on the time of exposure to the compound and that chronic effects on the liver cell turnover may play an important role in its ability to promote hepatocarcinogenesis.