Chapter 3: Phenotypic Characterisation of Melanotransferin Knockout Mouse and Down-Regulation of Melanotransferrin in Melanoma Cells
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CHAPTER 3: PHENOTYPIC CHARACTERISATION OF MELANOTRANSFERIN KNOCKOUT MOUSE AND DOWN-REGULATION OF MELANOTRANSFERRIN IN MELANOMA CELLS “An ignorant person is one who doesn't know what you have just found out.” Will Rogers, 1879 - 1935 This Chapter is published in: 1. Sekyere, E.O., Dunn, L.L., Suryo Rahmanto, Y., and Richardson, D.R. (2006) Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo. Blood. 107(7):2599-601. IF 2006: 10.4 *Selected by the Editors of Blood as a Plenary Paper which this journal describes as “papers of exceptional scientific importance”. 2. Dunn, L.L., Sekyere, E.O., Suryo Rahmanto, Y., and Richardson, D.R. (2006) The function of melanotransferrin: a role in melanoma cell proliferation and tumorigenesis. Carcinogenesis. 27(11):2157-69. IF 2006: 5.3 54 3.1. Introduction Melanotransferrin or melanoma tumour antigen p97 is an iron (Fe)-binding transferrin (Tf) homologue originally identified at high levels in melanomas and other tumours, cell lines and foetal tissues [Brown et al. 1981a; Brown et al. 1982; Woodbury et al. 1980]. Initial studies found MTf to be absent or only slightly expressed in normal adult tissues [Brown et al. 1981a], while later investigations demonstrated MTf in a range of normal tissues [Alemany et al. 1993; Rothenberger et al. 1996; Sciot et al. 1989]. Recent studies showed MTf to be expressed at higher levels in the brain and epithelial surfaces of the salivary gland, pancreas, testis, kidney and sweat gland ducts compared with other normal tissues [Richardson 2000; Sekyere et al. 2005; Sekyere et al. 2006]. Nonetheless, the highest levels of MTf are still found in melanoma cells, in particular the SK-Mel-28 melanoma cell line which expresses 3.0-3.8 x 105 MTf molecules per cell [Brown et al. 1981a; Brown et al. 1982]. The MTf molecule shares many properties in common with the Tf family of proteins, including the ability of isolated and purified MTf to bind Fe from Fe(III) citrate complexes [Baker et al. 1992; Brown et al. 1981b; Brown et al. 1982; Plowman et al. 1983; Rose et al. 1986]. These characteristics and the high expression of MTf in melanoma cells suggested that it played a role in Fe transport, possibly assisting these tumour cells with their increased Fe requirements [Richardson & Baker 1990; Richardson & Baker 1991b; Richardson 2000]. However, unlike Tf, MTf is bound to the membrane by a glycosyl-phosphatidylinositol anchor [Sekyere & Richardson 2000]. Previous in vitro studies demonstrated that, although MTf could bind Fe, it did not 55 efficiently donate it to the cell [Richardson & Baker 1991a; Richardson & Baker 1990; Richardson & Baker 1991b; Richardson 2000]. To date, the function of MTf in both normal and neoplastic cells remains unknown. Many roles have been proposed for MTf involvement in physiological and pathological processes, including transcytosis of Fe across the blood brain barrier, angiogenesis, cell migration, plasminogen activation, chondrogenesis, eosinophil differentiation and Alzheimer’s disease [Demeule et al. 2002; Moroo et al. 2003; Sala et al. 2002]. However, definitive evidence for the functional role of MTf is lacking. Melanotransferrin knockout (MTf -/-) mice were generated by targeted gene disruption to determine the role of MTf. This Chapter provides a comprehensive assessment of the phenotype of this animal which is important for defining the function of MTf. This investigation shows that lack of MTf expression had no significant effect on the growth, development, behaviour, or metabolism of MTf -/- mice compared with wild-type (MTf +/+) littermates. However, whole genome microarray analysis showed changes in the expression of genes such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein D (Apod) when comparing MTf -/- mice with MTf +/+ littermates. To assess the role of MTf in neoplastic cells, MTf expression was down-regulated by post-transcriptional gene silencing (PTGS) in SK-Mel-28 melanoma cells. This study also shows that down-regulation of MTf expression impaired cellular proliferation, DNA synthesis, cellular migration in vitro and tumour formation in vivo. 56 Collectively, the work described in this Chapter is the first to conclusively demonstrate that MTf has no role in Fe metabolism in normal and neoplastic cells. 57 3.2. Materials and Methods 3.2.1. Animals Animal work was conducted in accordance with the University of New South Wales Animal Ethics Committee Guidelines. All mice were housed under a 12 h light-dark cycle, fed routinely with basal rodent chow (0.02% Fe) and watered ad libitum. 3.2.2. Generation of MTf -/- mice MTf -/- mice were generated by homologous gene targeting in embryonic stem (ES) cells. The targeting vector was linearised and electroporated into 129/SvJ ES cells grown in medium containing G418 (Invitrogen). The ES cell clones targeted at both the 5’ and 3’ ends were used to generate chimeras. High-percentage chimeric males were mated with C57BL/6 females to obtain germ line–transmitting heterozygous offspring of mixed 129/SvJ x C57BL/6 background. To delete the neor-cassette, male heterozygote (MTf +/- neo+) mice were mated to B6-deleter females to obtain (MTf +/- cre+ neo-) offspring. MTf +/- cre+ females were crossed with C57BL/6 males to remove cre, generating MTf +/- mice free of neor and cre. These heterozygotes were intercrossed to generate MTf +/+ and MTf -/- littermates. 3.2.3. Isolation and Genotyping of Genomic DNA from Animal Tissues Three week old mice were tail-tipped (approximately 5 mm of tail per mouse) and the tissue placed in sterile tubes. The tissue was dissolved overnight at 55°C and genomic DNA isolated and purified for PCR analysis using the DNeasy® Tissue Kit (Qiagen) in accordance with the protocol provided by the manufacturer. 58 Genotyping was carried out by Southern analysis [Sambrook et al. 1989] and polymerase chain reaction (PCR) using genomic tail DNA. Briefly, the PCR reaction mixture contained 200 μM of each dNTP, 0.5 μM of each primer, 1.5 mM MgCl2, 1x PCR reaction mixture and 1.0 U Taq DNA polymerase (Invitrogen). The PCR cycles were consisted of 3 min at 94°C, followed by 30-40 cycles at 94°C (30 s), 55-60°C (1 min) and 72°C (1 min), with a single final extension period of 5 min at 72°C. The PCR product was analysed on a 1.5% agarose gel in 1x TAE buffer. 3.2.4. Serum chemistry, haematology and histochemistry Serum chemistry and haematological parameters were determined using a Konelab 20i analyser (Thermo-Electron Corporation, Finland) and Sysmex K-4500 analyser (TOA Medical Electronics Co., Kobe, Japan), respectively. The total Fe-binding capacity (TIBC) was calculated by adding the serum Fe level and the unbound Fe-binding capacity (UIBC). The Tf saturation was calculated as serum Fe level/TIBC x 100. Blood and bone marrow smears were prepared and stained with Giemsa. A wide variety of tissues were dissected, fixed in formalin, sectioned and stained with haematoxylin and eosin and Prussian blue. These were assessed by two independent veterinary pathologists. 59 3.2.5. Total tissue iron, copper and zinc determinations Total tissue Fe, copper (Cu) and zinc (Zn) concentrations were measured using inductively coupled plasma atomic emission spectrometry (ICP-AES) via standard techniques [Kingston & Jassie 1988]. 3.2.6. Microarray processing and analysis Six 17.5 week old female mice comprising four MTf -/- and two MTf +/+ from the same litter were used. Total RNA was isolated by grinding brain tissue in 1 mL of TRIzol® reagent (Invitrogen, Sydney, Australia). First strand cDNA synthesis was performed using a total of 15 μg of RNA by the Affymetrix One Cycle® cDNA synthesis kit and the cDNA products purified using the Affymetrix GeneChip® sample clean-up kit (Millenium Sciences, Victoria, Australia). Using the Affymetrix IVT® labelling kit (Millenium Sciences), biotin-labeled cRNA was prepared from the cDNA template. After further purification with the above mentioned clean-up kit, the quantity of product was ascertained using the Nanochip protocol on an Agilent Bioanalyser 2100 (Agilent Technologies, CA, USA). A total of 20 μg of labeled cRNA was fragmented to the 50-200 bp size range and the quality of the fragments checked again. Samples (cRNA, 0.05 μg/μL) that passed this checkpoint were then prepared for hybridisation to the mouse GeneChip® 430 2.0. The mouse GeneChip® 430 2.0 consists of greater than 39,000 transcripts and variants from over 34,000 well characterised mouse genes. On completion of hybridisation and washing, microarray chips were scanned with the Affymetrix GeneChip® Scanner 3000 (Millenium Sciences). Microarray data is available on the Gene Expression Omnibus 60 (http://www.ncbi.nlm.nih.gov/geo/) using the accession numbers GSM101412, GSM101413, GSM101414, GSM101415, GSM101416 and GSM101417. A two phase strategy was used to identify differentially expressed genes. First, genome- wide screening was performed using Affymetrix GeneChips®. The empirical Bayes procedure [Lonnstedt & Speed 2002] was applied in order to detect genes most likely to be differentially expressed between the MTf -/- and MTf +/+ samples. Individual p-values were then adjusted using the Holm step-down procedure to reduce the likelihood of false positives [Holm 1979]. Statistical analysis of data from the Affymetrix Genechips® was used to produce a list of thirty genes with the greatest fold change. This analysis was not meant to provide proof of differential expression. Rather, definitive evidence of differential expression was obtained from the RT-PCR assessment of samples used for the microarray analysis and also at least three other independent samples. 3.2.7. Construction of siRNA vectors, cell culture and transfections To prepare a construct capable of generating hairpin siRNA specific for human MTf mRNA, the pSilencer™ 3.1-H1 neo expression vector (Ambion, Texas, USA) was used.