Molecular cytogenetic analysis of chemoresistant non-Hodgkin's lymphoma patients with p53 abnormalities using fluorescence in situ hybridisation and comparative genomic hybridisation.

BACKGROUND: Alterations of the p53 gene at 17p13.1 as well as the gene for a transmembrane p-glycoprotein, ABCB1 (MDR-1) at 7q21.12, have been shown to be mostly associated with the phenomenon of multi-drug resistance (MDR) in human cancers. In order to better understand the mechanisms by which chemoresistance is mediated, non-hodgkin's lymphoma (NHL) patients overexpressing p53 mutant protein and resistant to CHOP chemotherapy, NHL patients without p53 overexpression and a Burkitt's lymphoma Raji cell line with p53 overexpression have been evaluated using fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). METHODS: Three chromosomes (1, 7, and 17) known to be associated with MDR and the presence of p53 mutant protein, were analysed by FISH. RESULTS: No obvious chromosomal aberrations such as translocations were found in any of the patients when compared to healthy individuals, which suggests that the three selected chromosomes might not be specifically related to NHL, with or without p53 overexpression. For CGH, gains and losses of chromosomal material have been identified and the changes were not only limited to the three selected chromosomes associated with MDR. A detailed analysis of the recurrent aberrations shows that most of the NHL patients have alterations on the chromosome arms 1p, 6q, 7q, 20q, 22q, and Xp, whereas patients with p53 overexpression predominantly show aberrations on 4p and 17q. CONCLUSION: Further characterisation of the genetic regions identified might more closely contribute to our understanding of acquired MDR in NHL. Alterations in the three evaluated chromosomes may be prevalent in other tumours. In the present study, using FISH and CGH, there was insufficient difference between NHL patients with and without p53 overexpression.

An in vitro model to study chemoresistance in non-Hodgkin's lymphoma patients over-expressing mutant p53.

INTRODUCTION: P-Glycoprotein plays a major role in regulating the concentration of chemotherapeutic agents inside the cytoplasm of normal and cancerous cells. The present in vitro study has primarily focused on the evaluation of the chemosensitising drug model, verapamil, as a P-glycoprotein antagonist not only to overcome chemoresistance in non-Hodgkin's lymphoma (NHL) chemoresistant cells (p53(+) i.e. over-expressing p53 mutant protein NHL cells) but also to evaluate and suggest the use of the single cell gel electrophoresis (SCGE) assay in the clinical setting. METHODS: Leucocytes from CHOP-chemoresistant NHL patients (p53(+) cells) were examined in the SCGE assay. Altered levels of DNA damage (tail moments) were determined by the assay not only in the leucocytes from clinical samples, but also in the Raji cell sub-lines (NHL model) which are also over-expressing p53 mutant protein. In addition, as a comparison, P-glycoprotein was examined in normal human leucocytes and Raji cells. RESULTS: Verapamil increased the tail moments induced by doxorubicin in all cell types over-expressing p53 mutant protein. DISCUSSION: The assay was successful for evaluating P-glycoprotein regulation. This suggests the applicability at the cellular level of the method as suitable for use in the clinical setting since it is reliable and could be used pre-clinically or perhaps instead of or alongside clinical trails. Cells from patients with a chemoresistant disease state or whose disease relapses subsequently could be treated with such novel experimental therapies in vitro to determine the necessity for the individual administration of a P-glycoprotein antagonist.

Sensitivity of different thalassaemia genotypes to food mutagens in the Comet assay.

Thalassaemia is a heterogeneous group of inherited anaemias, characterised by a reduction or total absence of one or more of the globin chains of haemoglobin. Individuals with thalassaemia major require regular blood transfusions in order to maintain their haemoglobin concentration at an appropriate level. An essential treatment in parallel with transfusions is iron chelation therapy to remove excess iron deposited in tissues from the transfused blood. The high iron levels in these patients make free oxygen radicals accessible, for example, through Fenton-type chemistry, and generate superoxide and hydroxyl radicals. Increased oxygen radical capacity is known to be associated with cancer and ageing. In a previous study, it has been shown that peripheral blood lymphocytes isolated from a sickle/beta thal double heterozygote-sickle phenotype thalassaemia patient, who was not undergoing chelation therapy, showed increased sensitivity to the effects of oxygen radicals and iron salts by comparison with lymphocytes from normal controls. Furthermore, in a later study, this patient also showed increased sensitivity to the dietary food mutagen 3-amino-1-methyl-5H-pyridol(4,3-b)indole (Trp-P-2) when compared to the control. The present study, therefore, investigated whether the above observation could be duplicated using different food mutagens in different thalassaemia genotypes. The effect of the food mutagens 2-amino-2-methylimidazolo(4,5-f)quinolone (IQ) and 2-amino-1-methyl-6-phenylimidazol(4,5-b)pyridine (PhIP) on lymphocytes of three different thalassaemia patients, a beta-thalassaemia major, a beta-thalassaemia/Hb E, and an alpha-thalassaemia trait with a 3.7-kb deletion, who were not undergoing chelation therapy were investigated using the Comet assay. All three thalassaemia genotypes showed increased sensitivity to both IQ and PhIP in comparison to the control, although with PhIP at the highest two concentrations (50 and 75 microM) the differences monitored with the alpha-thalassaemia trait were found not to be statistically significant (P > 0.05).

Modulation by flavonoids of the effects of a food mutagen in different thalassaemia genotypes in the Comet assay.

Thalassaemia is an inherited group of disorders caused by a reduction or total absence of one or more of the globin chains of the haemoglobin molecule. It has been shown that lymphocytes isolated from a sickle/beta thal double heterozygote-sickle phenotype patient showed increased sensitivity to the dietary food mutagen 3-amino-1-methyl-5H-pyridol(4,3-b)indole (Trp-P-2) when compared to the control. Furthermore, when a combination of Trp-P-2 with either quercitin or kaempferol was compared, the responses to Trp-P-2 were reduced to untreated control levels at the highest doses of quercitin and kaempferol. It has now been shown that using the food mutagens 2-amino-2-methylimidazolo(4,5-f)quinolone (IQ) and 2-amino-1-methyl-6-phenylimidazol(4,5-b)pyridine (PhIP) on lymphocytes of three different thalassaemia patients, a beta-thalassaemia major, a beta-thalassaemia/Hb E, and an alpha-thalassaemia trait with a 3.7-kb deletion, similar increased sensitivity could also be demonstrated. The present study investigated whether the modulatory effects of the flavonoids could be demonstrated in lymphocytes isolated from a beta-thalassaemia major and a beta-thalassaemia/Hb E patient. Lymphocytes from both a beta-thalassaemia major and beta-thalassaemia/Hb E patient showed increased sensitivity to PhIP when compared to the normal control. When a combination of PhIP and either quercitin or kaempferol was used, a reduction in the responses was seen, and at the highest doses of quercitin and kaempferol the responses were reduced to near untreated control levels and were significantly different when compared to PhIP alone (P < 0.05). It was concluded that lymphocytes from different thalassaemia genotypes showed increased sensitivity to different dietary food mutagens compared to normal lymphocytes and that flavonoids such as quercitin and kaempferol modulated the effects of these food mutagens in an antigenotoxic/antioxidant manner.