Gaetano Salvatore

Effect of Helicobacter pylori infection and its eradication on cell proliferation, DNA status, and oncogene expression in patients with chronic gastritis

BACKGROUND Helicobacter pylori, the main cause of chronic gastritis, is a class I gastric carcinogen. Chronic gastritis progresses to cancer through atrophy, metaplasia, and dysplasia. Precancerous phenotypic expression is generally associated with acquired genomic instability. AIM To evaluate the effect of H pylori infection and its eradication on gastric histology, cell proliferation, DNA status, and oncogene expression. METHODS/SUBJECTS Morphometric and immunohistochemical techniques were used to examine gastric mucosal biopsy specimens from eight controls, 10 patients withH pylori negative chronic gastritis, 53 withH pylori positive chronic gastritis, and 11 with gastric cancer. RESULTS All patients with chronic gastritis were in a hyperproliferative state related to mucosal inflammation, regardless of H pyloriinfection. Atrophy was present in three of 10 patients withH pylori negative chronic gastritis and in 26 of 53 with H pylori positive chronic gastritis, associated in 18 with intestinal metaplasia. DNA content was abnormal in only 11 patients with atrophy and H pylori infection; eight of these also had c-Myc expression, associated in six cases with p53 expression. Fifty three patients withH pylori positive chronic gastritis were monitored for 12 months after antibiotic treatment: three dropped out; infection was eradicated in 45, in whom cell proliferation decreased in parallel with the reduction in gastritis activity; atrophy previously detected in 21/45 disappeared in five, regressed from moderate to mild in nine, and remained unchanged in seven; complete metaplasia disappeared in 4/14, and markers of genomic instability disappeared where previously present. In the five patients in whomH pylori persisted, atrophy, metaplasia, dysplasia, and markers of genomic instability remained unchanged. CONCLUSIONS ChronicH pylori infection seems to be responsible for genomic instability in a subset of cases of H pylori positive chronic atrophic gastritis; eradication ofH pylori infection can reverse inflammation and the related atrophy, metaplasia, and genomic instability.

Differential immunohistochemical detection of transforming growth factor α, amphiregulin and CRIPTO in human normal and malignant breast tissues

The expression of growth factors, such as transforming growth factor α (TGFα), amphiregulin (AR) and CRIPTO, a type‐1 tyrosine‐kinase growth factor receptor‐(erbB‐2), and a tumor‐suppressor gene (p53), that have been implicated in the development and/or the progression of breast cancer, was evaluated by immunohistochemistry in 100 human primary infiltrating breast carcinomas (IBC). AR and CRIPTO immunoreactivity was also assessed in 55 human breast ductal carcinomas in situ (DCIS). Within the 100 IBC, 80, 50, 73, 17, and 34 tumors expressed moderate to high levels of TGFα, AR, CRIPTO, erbB‐2, and p53 respectively. In addition, AR and CRIPTO immunoreactivity were found in 11 and in 26 out of 55 DCIS respectively. In contrast, only 4, 3, and 2 out of 10 normal mammary‐gland samples were weakly positive for TGFα, AR, and CRIPTO expression, respectively, whereas none was positive for erbB‐2 or p53. Within the 100 IBC, expression of erbB‐2 significantly correlated with high histologic and nuclear grading, with high growth fraction, and with estrogen‐receptor(ER)‐ and progesterone‐receptor(PgR)‐negative tumors. A statistically significant correlation was also observed between p53 expression and high histologic grading, high growth fraction, and PgR‐negative tumors. In contrast, no significant correlations were found between TGFα, AR, and CRIPTO immunoreactivity and various clinicopathological parameters, with the exception of a positive correlation between TGFα and ER expression. These data demonstrate that TGFα, AR, and CRIPTO expression are significantly increased in malignant mammary epithelium relative to normal epithelium. In particular, the differential expression of CRIPTO may serve as a potential tumor marker for breast carcinogenesis.

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas

Galectin‐1 and galectin‐3 are galactoside‐binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional‐cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin‐1 mRNA levels were highly increased in most high‐grade tumors compared with normal bladder or low‐grade tumors. Western blot and immuno‐histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin‐1 in tumors. Galectin‐3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade. Int. J. Cancer (Pred. Oncol.) 84:39–43, 1999.

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-κB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKγ, increasing availability of IKKγ and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-κB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

Expression of COX-2, mPGE-synthase1, MDR-1 (P-gp), and Bcl-xL: a molecular pathway of H pylori-related gastric carcinogenesis.

Helicobacter pylori up‐regulates cyclo‐oxygenase‐2 (COX‐2) expression, which in turn is involved in tumourigenesis. Recently, a causal link between COX‐2 and multidrug resistance 1 (MDR‐1) gene expression, implicated in cancer chemoresistance, has been demonstrated. Thus, the expression of COX‐2 and the downstream enzyme involved in PGE2 biosynthesis, microsomal PGE‐synthase1 (mPGES1), was correlated with P‐gp, the product of MDR‐1, and the anti‐apoptotic protein, Bcl‐xL, in gastric biopsies from patients with H pylori infection and in patients with gastric cancer. In a retrospective analysis of endoscopic and pathology files, 40 H pylori‐negative patients (Hp−), 50 H pylori‐positive patients who responded to eradication therapy (Hp+R), 84 H pylori‐positive patients who did not respond to eradication therapy (Hp+NR), and 30 patients with gastric cancer (18 intestinal and 12 diffuse types) were selected. COX‐2, mPGES1, P‐gp, and Bcl‐xL were detected by immunohistochemistry. COX‐2, mPGES1, P‐gp, and Bcl‐xL expression was undetectable in gastric mucosa from Hp− patients. By contrast, COX‐2 and mPGES1 expression was detected in 42% and 44% of Hp+R patients, respectively, and in up to 66% (range 63–66%) of Hp+NR patients (p < 0.05). The expression of COX‐2 and mPGES1 correlated significantly (p < 0.0001) with that of P‐gp and Bcl‐xL. High levels of COX‐2, mPGES1, P‐gp, and Bcl‐xL expression were found in intestinal‐type gastric cancer samples. In conclusion, H pylori‐dependent induction of COX‐2 and mPGES1 is associated with enhanced production of P‐gp and Bcl‐xL that may contribute to gastric tumourigenesis and resistance to therapy. Copyright

PATZ1 gene has a critical role in the spermatogenesis and testicular tumours

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1−/− mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up‐regulation and mis‐localization could be associated to the development of TGCTs. Copyright

Clinical response, vascular change, and angiogenesis in gonadotropin-releasing hormone analogue-treated women with uterine myomas

Objective: Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) are involved in the pathogenesis of leiomyomas and influence angiogenesis, which is necessary for growth of leiomyomas. Gonadotropin-releasing hormone analogue (GnRH-a) treatment might modify the growth factor expression and the blood supply in myomas. We investigated the effects of GnRH-a treatment on some clinical parameters, on the immunohistochemical expression of bFGF, VEGF, and PDGF, and on the vasculature of leiomyomas. Methods: Thirty-one women were treated with leuprolide acetate for 3 months; 55 untreated patients formed the control group. Hematologic parameters were assessed at the admission, after GnRH-a treatment, and after surgery. Uterine volume was evaluated by ultrasonography. The immunoexpression of bFGF, VEGF, and PDGF and of the endothelial markers CD34 and CD105, as well as the vascular pattern, were studied in leiomyomas, comparing treated and untreated patients. Results: Hematologic parameters improved and uterine volumes decreased after GnRJH-a treatment. The immunoexpression of bFGF, VEGF, and PDGF decreased in treated myomas, together with the total number of vessels and the angiogenetic vessels. Conclusion: This study confirms the clinical response of uterine shrinkage after GnRH-a treatment. A pathogenetic role of bFGF, VEGF, and PDGF in myoma growth and vascularization is suggested. Finally, this study indirectly confirms the importance of the vasculature in leiomyoma growth.

Modulation of in vivo growth of thyroid tumor-derived cell lines by sense and antisense vascular endothelial growth factor gene.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT – PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Expression of teratocarcinoma-derived growth factor-1 (TDGF-1) in testis germ cell tumors and its effects on growth and differentiation of embryonal carcinoma cell line NTERA2/D1.

The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undifferentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to differentiating agents such as retinoic acid (RA) and hexamethylen-bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of different histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undifferentiated teratocarcinomas. To address the questions of how TDGF-1 (previously called CRIPTO) may affect the growth and/or the differentiation of embryonal carcinoma cells, we have characterized the effects of exogenous recombinant TDGF-1 protein on the proliferation rate and differentiation potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undifferentiated and differentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block differentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms.

Desmin‐free cardiomyocytes and myocardial dysfunction in end stage heart failure

Our aim was to evaluate the desmin content in the myocardial tissue of patients with end‐stage heart failure of ischaemic origin and to assess its role on cardiac function. We studied 18 explanted hearts from patients transplanted for end‐stage heart failure due to ischaemic cardiomyopathy (ICM). Control myocardial tissue was obtained from the cardiac biopsies of six women with breast cancer taken prior to commencing chemotherapy with anthracyclines, four male donors for heart transplantation and two autoptic hearts from patients who died due to non‐cardiac events. Myocardial tissue, obtained from the left ventricle (remote zone from infarcted area), was analyzed by light and confocal immunochemistry (desmin) microscopy. The desmin content of myocardial tissue was obtained by real‐time PCR. Cardiac function was evaluated by echocardiographic and right heart catheterization data, obtained before heart transplantation. Confocal microscopy evaluation showed a significant decrease in the number of desmin‐positive myocytes (P<0.01) in ICM hearts compared to controls. At real‐time PCR evaluation, there was a reduction (P<0.01) in desmin content in the ICM patients compared to controls. A negative correlation was found between desmin‐free cardiomyocytes and ejection fraction (EF) (r=−0.834; P<0.02) on echocardiogram. A negative relationship (r=−0.688) was also found between desmin‐negative myocytes and capillary wedge pressure. In conclusion, the myocardial tissue of patients with end‐stage heart failure of ischaemic origin, shows a decreased number in desmin‐positive myocytes at immunochemistry evaluation compared to normal individuals. This deficiency in cytoskeletal intermediate filament content is associated with reduced cardiac function.