Modifications of the radiosensitivity of a renal cancer cell line as a consequence of stable TIMP-1 overexpression.

PURPOSE: To investigate the potential effects of stable tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) overexpression on DNA damage and cell killing following low-dose gamma-radiation and whether this up-regulation interfered with the activation of the matrix metalloproteinase -2 (MMP-2) and -9 (MMP-9) in a highly metastatic renal carcinoma cell line. MATERIALS AND METHODS: Stable transfections were carried out using the cytomegalovirus expression plasmid pRc/CMV carrying TIMP-1 cDNA and LIPOFECTAMINE reagent. TIMP-1 expression in selected clones was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Exponentially growing Caki-1 cells were treated with sub lethal doses of ionizing radiation (0- 10Gy) either alone or following stable TIMP-1 transfection. DNA damage was assessed by the Alkaline Comet Assay and cell survival was determined by a clonogenic assay. Caki-1 cell cycle alterations following TIMP-1 transfection were assessed by fluorescence activated cell sorting (FACS) analysis of propidium iodide (PI)-stained cells. The interactions between TIMP-1 and MMP-2 and MMP-9 were analysed 24 hours post-irradiation by means of gelatin zymography. RESULTS: Three clones with varying degrees of TIMP-1 expression were selected and used for further analysis. TIMP-1 transfected Caki-1 cells displayed significantly higher mean tail moment values (p < 0.05) following irradiation at doses between 5 and 10 Gy relative to that seen with radiation alone. The TIMP-1 radiosensitizing effect was accompanied by large decreases in the survival fraction of the parental Caki-1 cell line and significant increases in the alpha-parameter of the linear-quadratic fit. These effects were directly correlated to the degree of TIMP-1 gene expression detected in the selected clones. Interestingly, elevated levels of TIMP-2 protein were detected in the three TIMP-1 clones compared to TIMP-2 levels present in Caki-l cells. The three clones also displayed marked phenotypic alterations relative to their parental cell line. Significant increases in the percentage of cells arrested in the G2/M phase of the cell cycle were detected in the three clones under normal growth conditions and reduced serum conditions (p < 0.05). When the TIMP-1 clones were assessed for their MMP-2 activity, a marked decrease in the MMP-2 mean protein levels was detected in clone T1-3 following irradiation at doses between 2 and 6 Grays (Gy) (p < 0.01) and clone T1-2 at 2- 5Gy (p < 0.05). MMP-9 activity was differentially affected by ionizing radiation in the three TIMP-1 clones. T1-3 and TI-2 displayed significantly reduced MMP-9 levels at various dose points whereas T1-1 exhibited elevated levels of MMP-9 activity at higher doses of treatment (p < 0.05). CONCLUSION: These results demonstrate a dual role for the TIMP-1 overexpression in this renal carcinoma cell line, both as radiosensitizing agents and effectors of MMP-2 and MMP-9 activity.

Annexin A2 expression during cellular differentiation in myeloid cell lines.

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on many cell types. It consists of a short hydrophobic tail (Ser(2)-Asn(32)), which dictates its function, and a core domain (Phe(33)-Asp(339)), which is involved in phospholipid binding. Annexin A2 has been implicated in a number of biochemical processes, including cell proliferation, foetal immune tolerance, ion-channel activation, cell-cell interactions and the bridging of membranes. Annexin A2 is reported to be a powerful activator of plasminogen and, therefore, is implicated in many normal and pathological processes such as haemostasis and metastasis. Myeloid cell lines are used, extensively, to study many aspects of cellular proliferation, differentiation and function. In the present study, we have used flow cytometry and real-time PCR to investigate the role of annexin A2 expression in the proliferation and differentiation of a number of myeloid cell lines. The results demonstrated that annexin A2 expression was affected when the cells were induced to differentiate by stimulation with all-trans-retinoic acid. Annexin A2 may, therefore, be an important player in cellular differentiation and its disorders.

Molecular, cytogenetic and genetic abnormalities in MDS and secondary AML.

Myelodysplasia (MDS) is a clonal disease, which increases with age, suggesting that multiple steps are required for the evolution of the condition. Approximately 30% of MDS evolve into acute myelogenous leukemia (AML). In this review, we intend to delineate the genetic events, which may drive this sequence and therefore we will focus primarily on cytogenetic abnormalities where the genes have been identified and oncogenes and suppressor genes that have been implicated. In terms of the biological mechanisms, which characterise this process, it is generally thought that the MDS cell has impaired differentiation, and has increased apoptosis. As the disease progresses in addition, the cells have increased proliferation. As the disease evolves, the population of cells, which predominate remain immature, have decreased apoptosis and in many cases, upregulate anti-apoptotic genes and have deregulated proliferation as the number of blast cells increase. Etiological factors, which contribute to the development of leukemia, include therapeutic agents administered for a primary malignancy. The cytogenetic abnormalities, predisposition factors and genes involved in secondary leukemia will also be reviewed.

Dietary supplementation with the anti-tumour promoter quercetin: its effects on matrix metalloproteinase gene regulation.

Dietary modification, especially the consumption of larger amounts of fruits and vegetables can act to decrease the risk of a variety of human cancers. Quercetin, a bioflavonoid widely distributed in fruits and vegetables has been shown to have a chemoprotective role in cancer, through complex effects on signal transduction involved in cell proliferation and angiogenesis. In this study we examined the effects of dietary supplementation of quercetin (30 mg per day) incorporated into a black currant drink. Healthy male subjects aged between 33 and 64 years (mean=47.1 years) received either quercetin or placebo for 14 days. Blood samples were taken at baseline and upon completion of the study and analysed for full blood count, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinse-1 and -2 (TIMP-1 and -2) plasma levels using ELISA techniques. RNA was extracted from the peripheral blood lymphocytes and reverse transcriptase-polymerase chain reaction (RT-PCR) carried out for MMP-2 and TIMP-1, TIMP-2 gene expression determination. Supplementation of the diet with quercetin did not alter the MMP-2 or TIMP-2 gene transcription or plasma protein levels of the healthy subjects in this study. The TIMP-1 gene transcription and plasma protein levels (311+/-70 ng/ml at baseline to 183+/-35 ng/ml post-supplementation, P<0.05) of the subjects in this study were, however, significantly decreased following quercetin supplementation. This is an interesting result, as there is some controversy over the functions of TIMP-1 in tumour progression. In certain model systems, artificially increased TIMP-1 levels prevent or decrease tumour growth. However, in other studies high levels of TIMP-1 have been correlated with aggressive disease and poor prognosis in patients with certain malignancies. This study has outlined a potential role for the anti-tumour promoter quercetin as a dietary mediator of the carcinogenic cascade.

Phenotypic alterations in Caki-1 cells as a consequence of TIMP-1 overexpression.

Maintenance of the extracellular matrix (ECM) is important for tissue integrity and cellular physiology. Normal ECM turnover is regulated by a balance between matrix metalloproteinases and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). In metastasis, this balance favours increased ECM degradation. The objective of this study was to determine the effects of TIMP-1 overexpression on the metastatic process. To this end, we stably transfected a renal carcinoma cell line, Caki-1, with TIMP-1, using a pRc/CMV expression plasmid and LIPOFECTAMINE transfection reagent. The resultant clones displayed increased adhesion on the ECM substratum, including collagen type IV and laminin, and altered invasive capacity through fibronectin and Matrigel, dependent upon the level of TIMP-1 expression. These changes were not due to altered integrin expression, as assessed by flow cytometry. As well as protease inhibitory activity, TIMPs can influence cell proliferation and cell survival. The TIMP-1 clones displayed no changes in proliferation under normal growth conditions, compared with Caki-1 cells. However, under reduced serum conditions, the TIMP-1 clones had a greater percentage of cells in both S (P<0.05) and G(2)/M (P<0.005) phases and less cells in G(0)/G(1) (P<0.001) of the cell cycle than Caki-1 cells. The results confirm a dual role for TIMP-1 in invasion and metastasis, and provide further clues behind the molecular mechanisms in these processes.

Bioavailability of n-3 polyunsaturated fatty acids (PUFA) in foods enriched with microencapsulated fish oil.

OBJECTIVE: Incorporation of fish oil into food products provides a means of increasing n-3 fatty acid intake, particularly in populations where fish consumption remains low. The aim of the present study was to evaluate the bioavailability of n-3 PUFA in microencapsulated fish-oil-enriched foods compared with an equal amount of n-3 PUFAs contained in fish oil capsules. METHODS: Twenty-five healthy female volunteers were randomly assigned to one of two groups for the 4-week intervention: one group received 0.9 g of n-3 PUFA/day as fish oil capsule (capsule group), while the second group (food group) received an equal amount of n-3 PUFA/day from enriched foods. Baseline and post-intervention samples were analysed for platelet fatty acid composition. RESULTS: There was no significant difference in the change in platelet arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) between the two groups following the intervention. CONCLUSIONS: The results indicate that n-3 PUFA from microencapsulated fish-oil-enriched foods are as bioavailable as n-3 PUFA in a capsule. Fortification of foods with microencapsulated fish oil, therefore, offers an effective way of increasing n-3 PUFA intakes and status in line with current dietary recommendations.

The effect of low-dose fish oil supplementation on serum growth factors in healthy humans.

OBJECTIVE: To examine the effect of low-dose fish oil supplementation on specific growth factors, purported to play a central role in lesion formation, and also on the total growth factor activity of serum, as assessed by the induction of DNA synthesis in cultured human arterial smooth muscle cells. DESIGN: Randomized placebo-controlled double-blind intervention study. SETTING: Free-living population. SUBJECTS: Sixty-three healthy volunteers, 37 males and 26 females. INTERVENTIONS: Four treatment regimes with subjects receiving 0, 0.3,0.6 or 0.9 g/day of n-3 PUFA for an 8 week period. Blood samples were taken at baseline and following the 8 week intervention. All samples were analysed in batch following completion of the study. RESULTS: Consumption of fish oil had no effect on serum platelet-derived growth factor (PDGF), or transforming growth factor beta (TGFbeta) concentration. Furthermore, fish oil supplementation did not alter the total growth factor activity of serum. CONCLUSIONS: Results indicate that low-dose fish oil supplementation, equivalent to about two portions of fatty fish per week and providing less than 1 g n-3 PUFA/day, does not alter the levels of the major serum growth factors and does not modify total serum growth factor activity in healthy human volunteers. SPONSORSHIP: European Union shared cost project (FAIR-CT-95-0085).

Biological consequences of a point mutation at codon 969 of the FMS gene.

The FMS proto-oncogene encodes the cell surface receptor for colony stimulating factor-1 (CSF-1). Mutations of the FMS gene at codon 969, in the C-terminal region of the gene, have been detected in haematological malignancies. To ascertain the biological significance of a mutation at this codon, we have used a murine haematopoietic cell line, FDC-P1, containing a mutation at codon 969 that results in a phenylalanine replacing a tyrosine. FMS 969 mutant cells and v-fms transfected cells conferred interleukin 3 (IL-3) independent stimulation of FDC-P1 cells, whereas cells transfected with a wild-type FMS construct required exogenous IL-3 for growth. FDC-P1 cells containing a FMS 969 mutation and v-fms transfected cells were tumorigenic in nude mice. Binding studies with radioidonated CSF-1 revealed saturable specific binding in FMS wild-type cells with a Km of 0.9 mM; however, mutant FMS-containing cells did not display saturation kinetics, but instead exhibited a linear relationship between ligand concentration and amount bound. Constitutive expression of FOS was detected in 969 mutant cells in the absence of exogenous CSF-1, a phenotype that was only inducible in wild-type cells in response to CSF-1. FOS and JUNB expression by v-FMS transfected cells showed a similar pattern to FMS wild-type cells. This mutation has been detected in patients with haematological malignancies, and illustrates that the pathway of FMS 969 phenylalanine mutations and v-fms induced pathogenesis can be distinguished. These data indicate that there is a biological role for FMS codon 969 phenylalanine mutation which results in transformation of FDC-P1 cells.

Allelic loss of the FMS gene in acute myeloid leukaemia.

The FMS proto-oncogene encodes for the colony stimulating factor-1 receptor expressed on monocytes and B lymphocytes within the peripheral blood system. Allelic loss of the FMS gene occurs in patients with refractory anaemia and the 5q- syndrome associated with the myelodysplastic syndromes. To determine the frequency of FMS gene loss in patients with myeloid malignancy, 50 DNA samples from patients with acute myeloid leukaemia (AML) and 30 samples from haematologically normal samples were analysed using a quantitative Southern blotting technique. Allelic loss of one allele (hemizygous) was detected in five of 18 samples of AM-M4 and eight of 27 samples of AML M1, M2 and M3. In addition, loss of both FMS alleles (homozygous) was demonstrated in three of 18 samples of AML M4 and 0127 samples of AML M1, M2 and M3. One patient with AML M5 and one with AML M6 were assessed although no allelic loss of FMS was detected. Three samples from patients with secondary AML were also analysed and hemizygous loss was detected in one case. Homozygous or hemizygous loss of FMS was not detected in any of 30 DNA samples isolated from haematologically normal individuals. These data indicate that loss of the FMS gene is common in AML, with an increased frequency in those patients with AML subtype M4.

FMS mutations in patients following cytotoxic therapy for lymphoma.

Point mutations at codons 301 and 969 of the FMS proto-oncogene have been reported in both myelodysplasia (MDS) and acute myeloid leukaemia (AML). We report here the incidence of such mutations in patients at risk of developing secondary MDS and AML. Peripheral blood DNA from 70 patients in remission from lymphoma was screened for mutations by oligonucleotide (ONH) using mutant specific probes. Codon 969 mutations were detected in 11 of the 70 (15.7%) cases. No codon 301 mutations were detected. Five of these mutations were confirmed using an independent technique (single nucleotide primer extension analysis, SNPE) and a further mutation was detected in a single patient using single-stranded conformational polymorphism analysis (SSCP). No codon 969 mutations were detected in 62 lymphoma biopsy specimens from these patients or from three patients with detectable FMS mutations where pre-therapy marrow was investigated by ONH. No mutations at either codons 301 or 969 were detected by ONH in 61 normal controls. Somatic mutations at codon 969 of the FMS gene occur commonly following cytotoxic therapy for lymphoma and their detection indicates the presence of a clonally expanded population of abnormal cells.

RAS and FMS mutations following cytotoxic therapy for childhood acute lymphoblastic leukaemia.

Patients who have received cytotoxic therapy for primary neoplastic disease are at an increased risk of developing secondary (therapy-related) acute myeloid leukaemia (AML) or myelodysplasia (MDS). RAS and FMS mutations have been observed in patients with AML and MDS. It has been suggested that the mutational status within these genes may be predictive of early secondary leukaemic disease. In this study we have screened 50 haematologically normal patients in complete remission from childhood acute lymphoblastic leukaemia (ALL) for activating point mutations in the RAS and FMS proto-oncogenes. Such patients may be considered at risk of therapy-related disease. Codons 12, 13 and 61 were screened in RAS and codon 969 in FMS using the polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH). Three of the 50 patients (6%) were found to harbour N12 RAS mutations. One of these three patients (2%) had both a N12 RAS and FMS 969 mutation. Upon sequencing the RAS mutations, substitutions of serine, cysteine and aspartic acid for glycine were identified. The FMS 969 mutation was also confirmed, by sequencing, as a histidine substitution. RAS mutations were not detected in presentation samples indicating that these lesions have been somatically acquired presumably subsequent to cytotoxic therapy for the primary disease. Continued follow-up of these patients may indicate a role for these mutations in the development of secondary malignancies.

A C-terminal FMS mutation in a patient with B-cell malignancy.

The FMS proto-oncogene encodes a polypeptide growth factor receptor expressed on the cell surface of monocytes and B lymphocytes within the haematological system. Mutations of the FMS gene at codons 301 and 969 have been detected in a number of haematological disorders. Mutations at these codons are thought to be important in the pathogenesis of leukaemia in cells expressing a mutant receptor. Following our finding that the colony stimulating factor-1 receptor (CSF-1R) was expressed on B cells, we have assessed DNA from 17 patients with B-cell chronic lymphocytic leukaemia (CLL), 15 with acute lymphoblastic leukaemias (ALL), two samples from patients with B-cell non-Hodgkin's lymphoma (B-NHL), and 20 haematologically normal individuals for the presence of C-terminal mutations of the FMS gene. Using single stranded conformational polymorphism analysis (SSCP), a single band shift was detected resulting from a nucleotide insertion at codon 965 in the DNA isolated from a patient with B-NHL. These results indicate that mutations of the FMS gene in this region are rare in B-cell malignancy but may contribute to the pathogenesis of leukaemias and lymphomas in a small subset of patients. However, the presence of other mutations not detected using this type of analysis cannot be excluded.

In vitro mechanisms and models of gentamicin nephrotoxicity.

Nephrotoxicity is the dose limiting feature of gentamicin adminstration. The nephrotoxic potential of gentamicin was investigated in primary cultures of rat renal proximal tubular cells and in the established renal epithelial cell lines, LLC-PK(1) cells and MDCK cells. Polyaspartic acids have been reported to ameliorate the nephrotoxicity of gentamicin in vivo. However, these compounds have been reported to exert other toxic side effects. We investigated the role of magnesium-l-aspartate-hydrochloride in nephroprotection against gentamicin in these models of nephrotoxicity. Gentamicin admistration resulted in a reduction in the DNA and protein content of the primary proximal tubular cells and an impaired calcium uptake into these cells. Magnesium-l-aspartate-hydrochloride significantly reduced the extent of [(3)H]gentamicin binding to these cells. Transepithelial resistance determination in the established renal cell lines revealed that gentamicin disrupted intercellular communications, while magnesium-l-aspartate-hydrochloride abolished this gentamicin-induced alteration. The actions of gentamicin were shown to occur prior to any loss of cell viability as assessed by flow cytometry. It is concluded that these systems provide useful models for the investigation of gentamicin nephrotoxicity and that magnesium-l-aspartate-hydrochloride may be useful in ameliorating gentamicin nephrotoxicity in clinical conditions.