Detection of methylation in the CpG islands of the p16INK4A, RASSF 1A and methylguanine methyltransferase gene promoters in pancreatic adenocarcinoma.

Pancreatic cancer consists of an accumulation of genetic and epigenetic alterations. Recently, aberrant methylation of CpG islands of cancer-related genes has emerged as an important epigenetic mechanism of their transcriptional dysregulation during tumour development [1]. Therefore, new diagnostic methods, for early detection based on a better understanding of the molecular biology of pancreatic cancer, are required. We examined the methylation status of p(16INK4A), RASSF 1A and methylguanine methyltransferase (MGMT) genes considered to be inactivated by promoter methylation in several tumours.The p(16INK4A) is an important G1/S cell cycle regulator gene [2]. RASSF 1A gene is involved in apoptotic signalling, microtubule stabilization and cell cycle progression [3]. The MGMT gene removes mutagenic and cytotoxic alkyl-adducts from the O6-position of guanine in DNA. Hypermethylation of the gene leads to the inactivation of DNA repair and to microsatellite instability [4].To date, little is known about the exact role of hypermethylation of these genes in pancreatic adenocarcinoma, as the molecular mechanisms underlying these neoplasms remain poorly understood.

Preclinical studies of steroid-linked nitrosoureas in murine pancreatic adenocarcinoma PANO2.

PURPOSE: In earlier studies, this laboratory carried out research on the synthesis and anticancer evaluation of hybrid compounds, which combine two molecules in one such as homo-aza-steroidal esters (HASE) of carboxylic derivatives of N, N-bis (2-chloroethyl) aniline. In this combination, steroidal hormones are employed as carriers for transporting the alkylating agents to specific targeted tissues. Aiming to continue our research, we used alkylating agents, as nitrosoureas, instead of nitrogen mustards. In this work the N-[N- (2-chloroethyl)-N-nitroso-carbomoyl]-L-alanine (CNC-ala) has been used and was bound to 7 newly synthesized modified steroidal esters (carrier molecule) of nitrosourea and the hybrid molecules were tested for antitumor activity against PANO2 murine pancreatic adenocarcinoma. MATERIALS AND METHODS: PANO2 adenocarcinoma was used in this study. C57Bl mice were used for chemotherapy evaluation. The activity was assessed from the inhibition of tumor growth and the oncostatic parameter T/C %. RESULTS: The antitumor activity displayed by 7 hybrid steroidal esters of nitrosourea was quite interesting. It was able to discern 4 of 7 compounds that exhibited considerable antitumor activity, increasing the lifespan of the tumor-bearing mice by inhibiting the tumor growth. CONCLUSION: The comparative study of 7 newly synthesized hybrid steroidal esters of nitrosourea shows that the antitumor effects of compound 7, which has an enlarged (7 carbon atoms) A-lactamic ring and nitrosourea esterified at the position 17, which seems to be the most appropriate for the connection of a DNA cross-linking amino acid derivative is superior.

Cytotoxic effects of a mixed ligand copper(II) chelate complex against a panel of human and murine cancer cells in vitro. Theoretical study of the mechanism of biologic action through molecular modelling.

PURPOSE: In an effort to discover new compounds with anticancer activity, we have developed a novel copper (II) [Cu(II)] chelate complex with a tridentate ONNSchiff base ligand and the anion of salicylate and we evaluated the in vitro chemosensitivity of various human and murine tumor cell lines by measuring cell growth inhibition. The ultimate goal was to evaluate the existence of a potential antitumor activity of this complex. Beyond the cytotoxic activity assessment of the complex, we aimed at the elucidation of the underlying mechanism of action of this complex and its interactions with biological molecules, carrying out theoretical (quantum-chemical) calculations. MATERIALS AND METHODS: Cells grown in adherence or in suspension in 96-well microplates were exposed to Cu(II) complex for 24, 48 or 72 h. In vitro drug cytotoxicity was assessed by SRB and XTT colorimetric assays. Molecular modelling tools were used applying semiempirical and ab initio calculations. RESULTS: A series of experiments was carried out, showing a potent cytotoxic activity against most of the tested cancer cell lines. Quantum-chemical calculations demonstrate that the mechanism of the cellular damage can be explained, at least in part, by the ability of the nucleobases and nucleotides to be subject to nucleophilic attack on copper. CONCLUSION: Profound growth inhibitory effects were observed for the tested Cu(II) complex. It was also verified the hypothesis that the mechanism of action of this complex involves binding to DNA and RNA. These findings prompt to search for possible interaction of this complex with other cellular elements of fundamental importance for cell proliferation.

Histological and ultrastructural study of the effect of chemotherapy with 5-fluorouracil on normal liver of Wistar rats.

BACKGROUND: In this study, we analysed the histopathological and ultrastructural effects of systemic chemotherapy with 5-fluorouracil (5-FU) on the liver of healthy rats. METHODS: Twenty inbred Wistar rats were used as controls and another 20 rats received a bolus infusion, via the inferior vena cava, of 375 mg/m(2) 5-FU. The animals of each group were sacrificed 16 h, 3 days or 1, 2, 3 and 6 months postoperatively. RESULTS: Histologically, in one rat 16 h after 5-FU administration, we observed only a small necrotic focus of hepatocytes. Ultrastructurally, a mild vacuolization of cytoplasm, a decrease in mitochondria and an increase in lysosomes were observed. These abnormalities were restored 1 month later. CONCLUSION: Our results confirm that the particular dose, route and duration of 5-FU administration do not cause hepatotoxic morphological changes in the liver of healthy Wistar rats.

Effect of navelbine on inhibition of tumor growth, cellular differentiation and estrogen receptor status on Lewis lung carcinoma.

We studied the antitumor activity of Navelbine (NVB) together with its ability to induce cellular differentiation and to influence estrogen receptor status of Lewis lung carcinoma (LLC). A total of 32 C(57)B1 mice divided into 5 groups were used for transplantation of LLC. Four groups of mice were treated with 5.0, 2.5 and 1.25 mg/kg/day from day 1 to 9 and 1.25 mg/kg on days 1, 7 and 13. Eight mice were controls. The dose of 1.25 mg/kg/day was the most effective and produced 72.7% inhibition of tumor growth. Ultrastructurally, on day 1 the cells showed poor differentiation. On day 14, in the case of 72.7% inhibition of tumor growth the study revealed a significant restoration of the morphology of the cells. Estrogen receptors gave a positive value in contrast to the initial measurement which was negative. The present study demonstrated good antitumor activity of NVB on LLC. NVB may also induce cellular differentiation and influence the estrogen receptor status.

A new approach in the management of urothelial tumors using GM-CSF on marker lesions: an ultrastructural and immunohistochemical study on the macrophage population in bladder mucosa.

Our purpose was to investigate a new therapeutic model, GM-CSF-targeted immunomodulation on transitional cell carcinoma (TCC) marker lesions and to evaluate the immunologic response of the bladder mucosa. Eleven patients with pTa or pT1 bladder cancer were eligible for the study. All lesions were removed by transurethral resection (TUR) except for a marker lesion. All patients received 8 weekly instillations of 300 microg of GM-CSF, after which cystoscopy with bladder biopsies +/- TUR was repeated on adjacent urothelium or tumor or both. Paraffin-embedded sections were immunohistochemically stained with CD68, which labels monocytes and macrophages. The CD68+ cell population was evaluated as 1+ to 3+. Comparable specimens were routinely processed for ultrastructural analysis. Complete response was observed in 6 patients (55%), persistent tumor occurred in 4 patients (approximately 36.4%), and 1 patient (8.6%) showed recurrence. Immunohistochemically, an at least twofold increase in the number of the CD68+ cells was observed in all responders. Submicroscopically, migration of macrophages to the surface layer occurred. Macrophages showed an extensive lysosomal system and pseudopodia. This study indicates that the prophylactic treatment of TCC with GM-CSF may induce immunomodulatory effects on macrophage activities, which could be associated with the clinical evolution of the disease.

Natural killer (NK) cell assay within bladder mucosa of patients bearing transitional cell carcinoma (TCC) after interferon (IFN) therapy: an immunohistochemical and ultrastructural study.

The authors studied the number and the ultrastructural evidence of NK cell activation in the non-involved urothelium in patients with transitional cell carcinoma (TCC) of the urinary bladder, before and after transurethral resection (TUR) and interferon (IFN) therapy. Eight male patients, free of recurrence 1 year after TUR and IFN-a2b therapy, were studied. Each patient received 22 instillations of 50MU of IFN-a2b over a period of 1 year. Two specimens from the non-involved urothelium, one adjacent to and another away from the tumour, were obtained before and after therapy, for immunohistochemical and ultrastructural studies. The number of NK cells was evaluated immunohistochemically in paraffin sections with the CD57 monoclonal antibody, and their activation was detected by routine electron microscopy processing. Before treatment, few NK cells were randomly found in the lamina propria. At the end of therapy, however, their number increased and NK cells were found to infiltrate the urothelium, a finding that was not observed before treatment. The number of NK cells did not correlate with the degree of the inflammatory infiltrate of the mucosa. Moreover, the ultrastructural study revealed activation of NK cells with enhanced cytolytic activity. IFN therapy increases the number and promotes the activation of NK cells within the bladder mucosa. This finding could be of clinical significance in the prevention of tumour recurrence, given that NK cells enhance the immunological defense mechanisms of the bladder.

Correlation of apoptosis with the distribution and composition of lymphocytic infiltrate in human breast carcinomas.

We studied the lymphocytic infiltrate in human breast carcinomas and correlated its distribution and composition with the apoptotic index of cancerous cells. Paraffin sections from 36 human breast carcinomas with prominent lymphocytic infiltration were immunohistochemically stained with L26, UCHL1 and Leu-7 antibodies. Apoptotic index was expressed as the number of cells undergoing apoptosis per 20 HPF. The lymphocytic infiltrate was composed mainly of T-cells, usually peripherally but in 17 cases intratumorally distributed. T-cells, in contact with apoptotic cells, were observed in 11 cases. B-cells formed small aggregates at the periphery of the tumor. NK cells were present isolated in the infiltrate. No correlation was found between the type and distribution of the lymphocytic infiltrate and apoptotic index. The presence of intratumoral T-lymphocytes, occasionally observed in contact with apoptotic cells, may suggest that they could be partly associated with the apoptotic death of cancerous cells.

Ultrastructural study of the urothelial lysosomal system in patients with transitional cell carcinoma after transurethral resection and interferon therapy.

The epithelial cells of human bladder urothelium contain a prominent lysosomal system on the surface layer which involves autophagic, phagocytotic and excretory processes. The noninvolved urothelium of tumor-bearing patients, however, does not contain this well-developed lysosomal system. Interferon restores the differentiation of the urothelium. Its action on the lysosomal system, however, has not been investigated. We studied ultrastructurally the noninvolved urothelium of eight patients with transitional cell carcinoma who after transurethral resection and intravesicular interferon instillations for 2 years did not develop recurrence. We noted that the number and size of lysosomes increased, being most numerous within the cells of the surface layer. Characteristic large lysosomes with the morphology of multivesicular bodies were also evident. These multivesicular bodies were almost entirely filled with small vesicles containing a dense core. Our findings show that after 2 years of interferon administration a re-appearance of a highly developed lysosomal system of the noninvolved urothelium was evident. This restoration to the normal morphology with reappearance of the lysosomal system, which could be partly attributed to interferon therapy, may be of clinical significance for prevention of tumor recurrence.

Ultrastructural study of the noninvolved urothelium of tumor-bearing patients after two years of intravesical interferon therapy.

OBJECTIVE: Seventeen patients with transitional cell carcinoma of the urinary bladder were studied. Twelve patients did not have a recurrence 2 years after a transurethral resection (TUR) followed by interferon (IFN)-alpha 2b treatment. This observation led us to study the ultrastructural morphology of the noninvolved urothelium in 8 of the above 12 patients. METHODS: All patients had a primary solitary grade I or II tumor. Topical therapy was started after TUR. Each patient received a total of 22 instillations of 50 MU IFN-alpha 2b in 12 months according to the standard procedure. After the first year, a repetitive dose of 50 MU IFN-alpha 2b was administered every 2 months for a period of 1 more year. RESULTS: At the end of therapy, certain ultrastructural modifications were observed indicating a partial restoration of the urothelium: the existence of asymmetric unit membrane, a well-developed Golgi apparatus and an increase of the filaments. The cells were joined to each other with well-developed tight junctions. Tubuloreticular inclusions were also observed. CONCLUSIONS: Prevention of recurrence by restoring the morphology of the noninvolved urothelium in response to IFN treatment deserves further examination.

Natural killer cell activation after interferon administration in patients with metastatic renal cell carcinoma: an ultrastructural and immunohistochemical study.

OBJECTIVE: We studied the intralesional number of natural killer (NK) cells and the ultrastructural evidence of their activation in patients with metastatic renal cell carcinoma, after preoperative interferon (IFN) administration. METHODS: Tumor sections from 10 patients with metastatic renal cell carcinoma who received preoperative IFN (5 MU x 3 days) and from 10 patients with renal cell carcinoma were obtained. The number of NK cells was estimated immunohistochemically in paraffin sections with the CD57 monoclonal antibody and their activation was evaluated ultrastructurally by routine electron microscopy processing. RESULTS: The number of NK cells was increased in the tumors of the IFN-administered patients (5.3 cells vs. 1.6 in the controls, p = 0.03) while their ultrastructural features revealed activation and enhanced cytolytic activity, through facilitation of granule content release. CONCLUSIONS: IFN promotes the aggregation and activation of NK cells within the renal cell tumor, further strengthening the concept of its immunomodulatory activity.

The ultrastructure of the noninvolved urothelium of tumor-bearing patients before and after interferon treatment.

The ultrastructural morphology of the noninvolved urothelium of tumor-bearing patients before and after interferon-alpha 2b (IFN-alpha 2b) treatment was investigated. The ultrastructure of the normal bladder urothelium has been reported. The noninvolved urothelium of our patients already showed deviation from the normal. At the end of therapy, we noted a partial restoration of the urothelium to its normal morphology with the reestablishment of some of its normal features, i.e., a well-developed Golgi apparatus, the existence of parts of the asymmetric unit membrane, and the appearance of nuclear bodies. Prevention of tumor recurrence by restoring the ultrastructural morphology of the noninvolved urothelium with IFN-alpha 2b is a subject worthy of further investigation.

A new approach combining interferon and bromocriptine LA to prevent spontaneous mouse mammary carcinoma.

The development of spontaneous mammary carcinoma in C3H mice is mainly attributed to an oncogenic virus (MMTV) and the high prolactin levels of this inbred strain. Interferon (IFN) reduces the incidence of mammary carcinoma in virgin C3H mice when administered during lactation. Bromocriptine LA, an antiprolactin drug, also decreases the incidence of this tumor in C3H mice. The combined action of mouse IFN-alpha + beta and bromocriptine LA had a significant anticarcinogenic effect more significant than either of the above agents administered independently. This antitumor action was effective in virgin C3H mice only.

The influence of bromocriptine on the ultrastructure of cultured T47D (human breast cancer) cells.

Following a previous ultrastructural study of untreated T47D human breast cancer cells, we studied the influence of bromocriptine on the same cell line with the electron microscope. Our results suggest that bromocriptine exerts a direct effect on the morphology of T47D cells, which after treatment were better preserved. Some morphological evidence was noted hinting that colchicine and bromocriptine may have some similar properties. It is possible that the T47D cell line is biologically responsive to bromocriptine and therefore it is a potentially useful model for studies on the mechanism of action of bromocriptine on the morphological level in human breast cancer.

The effect of bromocriptine on the lactotroph cells in two inbred strains of mice C3H/Sy and AKR/Sy.

This paper deals with the effect of bromocriptine on the morphology and the number of lactotrophs in two inbred strains of mice, one with a high and the other with a low mammary cancer incidence. Bromocriptine decreases the number of lactotrophs in C3H mice while their number in AKR is not affected. These observations suggest that bromocriptine affects the two strains in a different way, probably due to their different prolactin levels in the pituitary. The above treatment resulted also in a shrinking of the RER and a reduction in size of prolactin granules, probably due to a decline in the secretory activity of lactotrophs. Of special interest was the appearance of a 'new' cell type with mixed characteristics of somatotrophs and lactotrophs. These findings, along with an increase in the number of chromophobes, suggest that bromocriptine can induce cell regression. Probably the 'new' cell is an intermediate form between chromophobes and lactotrophs and somatotrophs.

The influence of age of culture on the morphology of T47D cells.

The cell line T47D is extensively used as model in studying the role of hormones in human breast cancer. On the other hand the study of their morphology is largely incidental and does not take into account the age of culture. We studied therefore the morphology of T47D cell line with the electron microscope from the third day of culture to the seventh without adding new medium and serum to find out if the age of culture affects their characteristics. Our results suggest that there is a constant change of the cell morphology and architecture. Thus some cell organelles as the nucleus, tonofilaments, endoplasmic reticulum, mitochondria and intercellular secretory ductules are affected by the age of culture. Also we observed signs of degeneration, necrobiosis and even necrosis on the seventh day of culture. These observations suggest that the age of culture of these cells should be taken into account in experiment planning.

Effect of bromocriptine on the ovarian cycle of two inbred strains of mice.

Mice of the C3H/Sy (high incidence of spontaneous mammary cancer) and AKR/Sy (low incidence of spontaneous mammary cancer) inbred strains, which have different hormonal profiles, were injected daily with bromocriptine for 1 month. The treatment increased the duration of the ovarian cycle of the AKR/Sy mice, whereas that of the C3H/Sy mice was not affected. It is suggested that the effect of bromocriptine on the ovarian cycle depends on the concentrations of plasma progesterone reached in each strain of mouse.

The influence of bromocriptine on the ultrastructure of the biceps femoris muscle in mice.

Bromocriptine and other dopaminergic agonists drugs are used in Parkinson's disease. In this paper we have studied the ultrastructure of striated muscle of mice after bromocriptine treatment. There was a tremendous increase in the number and size of mitochondria, as well as a very notable increase in the cristae. Some ultrastructure changes were also noted at the neuromuscular junctions. An explanation has been attempted in the light of other investigations concerning the relationship of microtubules and bromocriptine on the one hand, and microtubules and mitochondria on the other.