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4 November 1999, Volume 18, Number 46, Pages 6357-6366
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Article
Absence of APOBEC-1 mediated mRNA editing in human carcinomas
Jobst Greeve, Heinrich Lellek, Frank Apostel, Katja Hundoegger, Akbar Barialai, Romy Kirsten, Sybille Welker and Heiner Greten

Medizinische Kernklinik und Poliklinik, Universitäts-Krankenhaus Eppendorf, Martinistrae 52, D-20246 Hamburg, Germany

Correspondence to: Jobst Greeve, Medizinische Kernklinik und Poliklinik, Universitäts-Krankenhaus Eppendorf, Martinistrae 52, D-20246 Hamburg, Germany

Abstract

The transgene expression of the catalytic subunit APOBEC-1 of the apo B mRNA editing enzyme-complex can cause hepatocellular carcinoma in mice and rabbits. It has been proposed that aberrant editing of mRNA may represent a novel oncogenic principle. This investigation aimed to define whether such aberrant hyperediting mediated by APOBEC-1 occurs in human carcinomas. Editing and hyperediting of apo B, NAT1 or NF1 mRNA was not identified in any of 28 resected tumor specimens, including hepatocellular, bile duct, gastric, colorectal, pancreatic adeno- and neuroendocrine, lung adeno-, medullary thyroid and breast carcinoma, soft tissue sarcoma and neuroblastoma. In most types of carcinoma, significant levels for full-length APOBEC-1 mRNA could not be detected. Low level expression of APOBEC-1 was found in colorectal and gastric carcinoma where most of the APOBEC-1 mRNA is inactivated by alternate splicing. The `auxiliary' components of the apo B mRNA editing enzyme-complex are missing in many tumors including colorectal and gastric carcinoma, but are highly expressed in hepatocellular, lung adeno- and breast carcinoma all of which lack APOBEC-1. Taken together, either APOBEC-1 or the `auxiliary' components of the apo B mRNA editing enzyme-complex or both are missing in human carcinomas resulting in the absence of mRNA editing. Currently, there is no evidence that aberrant editing mediated by APOBEC-1 contributes to the tumorigenesis of natural human carcinomas.

Keywords

mRNA editing; APOBEC-1; human carcinoma; apo B; NAT1

Introduction

mRNA editing, the specific posttranscriptional base change in mRNA, is a genetic principle that has attracted a lot of attention in recent years (Simpson and Thiemann, 1995; Scott, 1995). Besides the various forms of editing for RNAs encoded in the mitochondria of plants or kimetoplastid protozoa, two paradigms for mRNA editing of nuclear encoded genes have been described, editing of apolipoprotein (apo) B mRNA and editing of mRNAs for certain subunits of glutamate-gated cation channels (GluRs) (Powell et al., 1987; Sommer et al., 1991). Editing of GluR transcripts changes genomically encoded adenosines into inosines altering amino acid residues at functional important domains of the encoded ion channels (Mass et al., 1997). This editing is mediated by double-stranded RNA-dependent adenosine deaminases of which so far three different forms have been cloned (Maas et al., 1997).

Editing from cytidine to uridine at nucleotide position 6666 in the apo B mRNA creates a premature translational stop codon and leads to the generation of the carboxyterminal truncated apo B-48 (Chen et al., 1987; Powell et al., 1987). In humans and many other mammalian species, the apo B mRNA is completely edited in the intestine but remains unedited in the liver (Greeve et al., 1993). Besides in the small intestine, the apo B mRNA is edited in the human stomach, colon and kidney (Teng et al., 1990). In some mammalian species (dog, horse, rat, mouse), also the hepatic apo B mRNA is edited and the hepatic synthesis of apo B-48 results in low plasma concentrations of the atherogenic LDL that are derived exclusively from apo B-100 containing VLDL (Greeve et al., 1993). The importance of hepatic apo B mRNA editing for plasma lipoprotein concentrations is further demonstrated by mice deficient in apo B mRNA editing and in the LDL receptor which have extremely high LDL plasma levels, are very susceptible to atherosclerosis and closely resemble human familial hypercholesterolemia (Powell-Braxton et al., 1998).

The editing of apo B mRNA is mediated by a multicomponent enzyme-complex, designated apo B mRNA editing enzyme (Greeve et al., 1991; Smith et al., 1991). The catalytic subunit APOBEC-1 (apo B mRNA editing enzyme catalytic polypeptide 1) is the only component of this enzyme-complex that has been conclusively characterized (Teng et al., 1993). APOBEC-1 is a member of the cytidine deaminase gene family with a novel RNA binding motif and requires additional `auxiliary' components for efficient editing (Navaratnam et al., 1993, 1995; Yamanaka et al., 1995; Anant et al., 1995). APOBEC-1 is the only component of the editing enzyme-complex missing in the human or rabbit liver (Giannoni et al., 1994; Greeve et al., 1996). In mouse and rat, APOBEC-1 is expressed in the liver as the result of a second promoter which leads to tissue-specific exon use and alternate splicing in the 5' untranslated region of the APOBEC-1 mRNA (Nakamuta et al., 1995; Qian et al., 1997; Greeve et al., 1998). Adenovirus-mediated gene transfer of APOBEC-1 into the liver of normal or LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) rabbits induces editing of the hepatic apo B mRNA and results in a drastic reduction of circulating LDL levels (Greeve et al., 1996).

In APOBEC-1 transgenic animals, the hepatic overexpression of APOBEC-1 mediated by the promoter of apolipoprotein E causes hepatocellular dysplasia and carcinomas (Yamanaka et al., 1995). The overexpression of APOBEC-1 leads to additional `hyperediting' of multiple cytidines downstream of the natural editing site C6666 in the apo B mRNA, and to aberrant `hyperediting' of cytidine residues in other mRNAs (Yamanaka et al., 1996, 1997; Sowden et al., 1996). One of these other mRNAs, NAT1 (novel APOBEC-1 target 1), was identified by the `mooring' motif in the apo B mRNA required for editing by APOBEC-1 (Yamanaka et al., 1997; Shah et al., 1991). NAT1 has homology to the carboxy-terminal portion of the eukaryotic translation initiation factor eIF4G, and most likely is a translational repressor (Yamanaka et al., 1997). Hyperediting of NAT1 mRNA leads to a loss of NAT1 function, which was postulated to contribute to the oncogenesis in the APOBEC-1 transgenic animals (Yamanaka et al., 1997). Further evidence that mRNA editing may be involved in tumorigenesis comes from studies in malignant neurofibromas of patients with neurofibromatosis type 1 (NF1) (Cappione et al., 1997). The mRNA of the NF1 gene has been demonstrated to be edited from C to U at nucleotide position 2914, creating a premature stop translation codon (Skuse et al., 1996). The editing of NF1 mRNA was much higher in neurofibromatous tumors as compared to normal tissues and it was concluded that mRNA editing may contribute to the genetic inactivation of the NF1 gene in NF1 malignancies (Cappione et al., 1997). Whether the editing of NF1 is mediated by APOBEC-1 together with novel trans-acting factors remains to be conclusively established (Skuse et al., 1996; Cappione et al., 1997). The third line of evidence that mRNA editing may be involved in oncogenesis is derived from an investigation of the Wilms tumor susceptibility gene WT1 (Sharma et al., 1994). The mRNA of WT1 can be edited from U to C at nucleotide position 839, changing codon 280 from CTC for leucine into CCC for proline (Sharma et al., 1994). The editing of WT1 mRNA is tissue-specific, temporally controlled and functionally important as the edited form of WT1 protein is less efficient in transcriptional repression than the unedited form (Sharma et al., 1994).

This study addressed the question as to whether APOBEC-1 mediates potential oncogenic hyperediting in human carcinomas. Out of 28 resected tumor specimens, no apo B mRNA editing or aberrant hyperediting of apo B, NAT1 or NF1 mRNA could be identified. APOBEC-1 was found to be either not expressed or only at very low levels. The carcinomas which express low levels of APOBEC-1 demonstrated a loss of the `auxiliary' components required for mRNA editing. Thus, there is no evidence that mRNA editing by APOBEC-1 represents an oncogenic principle in human carcinomas.

Results

Expression of APOBEC-1 and apo B mRNA in human carcinomas

APOBEC-1 mRNA could not be detected by primary RT - PCR in any of 28 individual tumor specimens of primary or metastatic carcinomas. Nested PCR, however, detected APOBEC-1 mRNA in all colorectal carcinomas studied, in one out of three hepatocellular carcinomas, in two out of three gastric carcinomas, in one of two pancreatic adenocarcinomas and in one of two lung adenocarcinomas. APOBEC-1 mRNA was not detectable by nested RT - PCR in bile duct, breast, medullary thyroid or pancreatic neuroendocrine carcinoma, and undetectable in two out of the three hepatocellular carcinomas, in one of the three gastric carcinomas, in one of the two pancreatic and in one of the two lung adenocarcinomas.

Primary RT - PCR generated a clearly visible product for apo B mRNA in five colorectal carcinomas, in all three hepatocellular carcinomas, in both bile duct carcinomas, in both breast carcinomas, in one of the three gastric carcinomas, in the neuroendocrine carcinoma and in the medullary thyroid carcinoma. Nested RT - PCR identified apo B mRNA in four additional colorectal carcinomas and in the neuroblastoma. Altogether, 20 out of 28 tumors were positive for apo B mRNA using primary or nested RT - PCR.

Apo B mRNA editing in human carcinomas

The 20 RT - PCR products for apo B mRNA were analysed by primer extension assay for editing at the natural editing site C6666 (Figure 1). Substantial amount of editing (>1%) at this site C6666 could not be detected in any of the 20 tumors. The apo B RT - PCR products of two colorectal carcinoma were cloned and individual clones were analysed by primer extension assay. All 80 and 83 cDNA clones, respectively, were unedited and contained the genomically encoded C at nucleotide position 6666. The apo B mRNA of two colorectal, two hepatocellular and one gastric carcinoma was amplified from nucleotide position 6552 to 6864 by RT - PCR and cloned. Twelve individual clones of each tumor were sequenced: No sequence divergence could be detected in these 60 apo B cDNA clones. Notably, the potential sites (C6675, C6699, C6707, C6718, C6738, C6743, C6762, C6782, C6783, C6802, C6806, C6807, C6815, C6833, C6840 and C6851) for aberrant hyperediting of hepatic apo B mRNA as identified in the APOBEC-1 transgenic animals (Yamanaka et al., 1996) were entirely unedited in these five human carcinomas.

The NAT1 mRNA of the same five tumors was amplified by RT - PCR from nucleotide position 2260 - 2758, cloned and sequenced. This region of the NAT1 mRNA contains 28 presumptive editing sites as identified in APOBEC-1 transgenic animals (Yamanaka et al., 1997). At four of these sites, C/U editing can create a premature stop translation codon. No C/U base heterogeneity at any of these sites could be detected in a total number of 36 NAT1 cDNA clones, all of which contained the unedited cDNA sequence as reported by Yamanaka et al. (1997).

Expression and editing of NF1 mRNA in human carcinomas

As bona fide mRNA editing has been reported at nucleotide position 2914 of NF1 mRNA in various cell lines, and in tumors of patients with neurofibromatosis type I, the NF1 mRNA was amplified by nested RT - PCR in human carcinomas. Specific products for NF1 mRNA were generated in 10 out of 11 tumors (Figure 2a). Substantial amount of editing at nucleotide position 2914 could not be detected in any of these specimens using primer extension analysis (Figure 2b). To substantiate these findings, the RT - PCR products of the NF 1 mRNA from colon carcinoma, hepatocellular carcinoma and neuroblastoma were cloned. The sequence of five independent cDNA clones from each of these three tumors was identical to the sequence as deposited at the GenBank nucleotide sequence database. Notably, none of these cDNA clones was edited at nucleotide position 2914 from C to U and contained T at this position.

Alternate splicing of APOBEC-1 mRNA in human carcinomas

In normal small intestine and most human carcinomas two discrete forms of APOBEC-1 mRNA with slightly different sizes were detected by nested RT - PCR (Figure 3). The larger product contained the expected 273 bp, but in the smaller product (245 bp) a deletion of 28 bp encoded by exon 2 was found. Therefore, the 245 bp product represents alternatively spliced APOBEC-1 mRNA in which exon 2 is skipped as originally reported by Hirano et al. (1997). In pancreatic carcinoma, breast carcinoma and neuroblastoma only this 245 bp RT - PCR product of APOBEC-1 was detectable (Figure 3). No RT - PCR products were generated from medullary thyroid carcinoma and soft tissue sarcoma as demonstrated by hybridization with oligonucleotides specific for exon 2 or exon 3 (Figure 3).

In normal small intestine and colorectal, gastric, hepatocellular and lung carcinoma both the 245 bp and, to much lesser extent, the 273 bp RT - PCR products hybridized with the exon 3 specific oligonucleotide, VW2. In pancreatic adenocarcinoma, breast carcinoma and neuroblastoma only the 245 bp product hybridized to VW2 (Figure 3). Hybridization with oligonucleotide SK3 specific for exon 2 generated a strong signal in normal small intestine, a weaker signal in colonic and gastric carcinoma, and only faint signals in rectal, pancreatic, hepatocellular and lung adenocarcinoma. The ratio of the 245 bp RT - PCR to the 273 bp RT - PCR-product was calculated by densitometry: It increased from 1.3 in normal small intestine to 1.7 in colon carcinoma, 3.0 in rectal carcinoma, 1.9 in gastric carcinoma, 4.0 in pancreatic carcinoma, 2.7 in hepatocellular carcinoma, 33.0 in breast carcinoma, 48.2 in neuroblastoma and 5.3 in lung adenocarcinoma.

Quantification of the two APOBEC-1 mRNA forms in human carcinomas

The expression of APOBEC-1 mRNA in normal small intestine and 11 different carcinomas was quantitated by ribonuclease protection assay using an antisense APOBEC-1 *RNA that spans exon 2. Regularly processed APOBEC-1 mRNA should protect a 242 bp fragment, while exon 2 skipped APOBEC-1 mRNA should result in a protected fragment of 214 bp. Two major protected fragments of 214 and 242 nucleotides were generated using total RNA from normal small intestine, colonic carcinoma, rectal carcinoma and gastric carcinoma (Figure 4). These two protected fragments appeared as a double-band each. These double-bands for the protected RNA-fragments were observed in a total of four independent RNAse protection assays. The reason for this phenomenon remained elusive. No protected fragments could be detected in yeast tRNA and in total RNA from pancreatic and hepatocellular carcinoma, lung adenocarcinoma, medullary thyroid carcinoma and soft tissue sarcoma (Figure 4). In the samples for colonic, rectal and gastric carcinoma, the two smaller fragments were more intense than the two larger fragments. Hybridization of 50 mug total RNA to a beta-actin antisense RNA resulted in a protected fragment of very similar intensity in all 11 tumor samples (Figure 4). This band was less intense in normal small intestine, for which only 20 mug instead of 50 mug total RNA were used (Figure 4).

Transcriptional start sites and APOBEC-1 mRNA integrity in human carcinomas

5'RACE was used to determine the integrity of APOBEC-1 mRNA in colonic and gastric carcinoma. Using an exon 5 specific primer (SK9), the primary 5'RACE generated a weak product of 670 bp only in human small intestine, but not in colonic or gastric carcinoma (Figure 5, upper panel). Using an exon 4 specific primer (SK10) or an exon 3 specific primer (SK2), 5'RACE generated products of approximately 540 bp or 320 bp, respectively, in human small intestine and, to less extent, in colonic and gastric carcinoma (Figure 5, upper panel). Nested 5'RACE generated an identical pattern of products in human small intestine and colonic or gastric carcinoma (Figure 5, lower panel). These 5'RACE products were cloned and sequenced: Four out of 12 clones from normal small intestine, two out of 12 clones from colonic carcinoma, and one out of 12 clones from gastric carcinoma contained exon 2. Additional gross structural rearrangements or point mutations were not found. In small intestine and both tumor specimens, the 5'RACE products extended up to 25 - 30 nucleotides beyond the translational start site ATG, as reported previously (Hirano et al., 1997). In addition, 3'RACE did not identify base changes or structural rearrangement in the 3'end of APOBEC-1 mRNA (data not shown).

Coexpression of full-length and exon 2 skipped APOBEC-1 mRNA in human hepatoma HuH-7 cells

To exclude an interference of the presumptive 36 amino acid translation product (pp36aa) of exon 2 skipped APOBEC-1 mRNA with full-length APOBEC-1, both cDNAs were cotransfected in human hepatoma HuH-7 cells. In the first series of experiments, increasing concentrations of pSVL-pp36aa (0 - 1.6 mug) were cotransfected with a fixed amount (0.4 mug) of pSVL-hAPOBEC-1. pSVL-beta-Gal was added to each transfection to a total amount of DNA of 2.0 mug. Transfection of 1.6 mug pSVL-beta-gal and 0.4 mug pSVL-hAPOBEC-1 resulted in beta-galactosidase activity in 75 - 80% of the cells. Changes in the editing of apo B mRNA with increasing amounts of pSVL-pp36aa (0 - 1.6 mug) were not detected (Figure 6, left panel). In a second set of experiments, a bicistronic expression plasmid was constructed in which both the full length and the exon 2 skipped APOBEC-1 mRNA were expressed by the use of the internal ribosome entry site (IRES) of encephalomyocarditis virus. Transfection of 2.0 or 0.4 mug of pSVL-hAPOBEC-1-IRES-pp36aa induced editing of apo B mRNA to the same extent as 2.0 or 0.4 mug pSVL-hAPOBEC-1 (Figure 5, right panel).

Expression of the `auxiliary' components of the apo B mRNA editing enzyme-complex in human carcinomas

The `auxiliary' components of the enzyme complex besides APOBEC-1 were determined in S100 extracts of the various tumors using complementation with recombinant APOBEC-1. Extracts of baculovirus-infected Sf9 cells expressing APOBEC-1 did not exhibit in vitro editing activity (Figure 7, lane 1). Addition of Hela-cell extracts containing `auxiliary' components but no apo B mRNA editing activity reconstituted apo B mRNA editing activity (Figure 8, lane 2 - 4). None of the S100 extracts from ten different carcinomas had endogenous apo B mRNA editing activity (Figure 7). After supplementation with recombinant APOBEC-1, strong in vitro apo B mRNA editing activity was observed in the extracts of the lung adenocarcinoma, the breast carcinoma and of one of the two hepatocellular carcinomas (Figure 7). Less in vitro editing activity was reconstituted in extracts from the other hepatocellular carcinoma, from the rectal carcinoma and from the neuroblastoma (Figure 7). Addition of Sf9 cell extract containing APOBEC-1 did not generate in vitro editing activity in extracts of colonic, sigmoideal, gastric and pancreatic carcinoma (Figure 7). Addition of increasing amounts of Hela-cell extracts containing abundant amounts of `auxiliary' components did not reconstitute in vitro apo B mRNA editing activity in any of the tumor extracts, including colorectal and gastric carcinoma (data not shown). Addition of increasing amounts of S100 extract from colorectal carcinomas did not diminish the editing activity of Hela-extracts supplemented with recombinant APOBEC-1, ruling out that colorectal carcinomas express specific inhibitors for mRNA editing (data not shown).

Discussion

The observation that the transgenic hepatic expression of APOBEC-1 can cause hepatocellular carcinoma demonstrated that APOBEC-1 can act as a potent oncogene (Yamanaka et al., 1995). This investigation aimed to establish whether aberrant mRNA editing mediated by APOBEC-1 may contribute to natural tumorigenesis in human patients. Unexpectedly, editing of apo B, NAT1 or NF1 mRNA could not be detected in 28 human carcinomas studied, and APOBEC-1 was found to be either not expressed or only at very low levels that were further inactivated by alternate splicing. The `auxiliary' components of the apo B mRNA editing enzyme-complex besides APOBEC-1 are expressed in some, but notably not in gastrointestinal carcinomas that express APOBEC-1 to detectable levels. Complementation of extracts from colorectal or gastric carcinomas, with extracts from Hela-cells containing the `auxiliary' components, demonstrated that in gastrointestinal carcinomas the expression of APOBEC-1 is not sufficient to promote mRNA editing. Thus, compared to normal human tissues (Teng et al., 1990), mRNA editing mediated by APOBEC-1 is lost in human carcinomas.

The induction of hepatocellular carcinoma in APOBEC-1 transgenic animals has been postulated to be due to aberrant hyperediting of mRNAs for genes that are involved in the control of cellular growth and differentiation (Yamanaka et al., 1995, 1997). Aberrant hyperediting of NAT1, a putative translational repressor, may contribute to tumor development in the APOBEC-1 transgenic animals (Yamanaka et al., 1997). Editing of the natural site in apo B mRNA or at aberrant sites in apo B or NAT 1 mRNA, however, could not be detected in any of the tumors studied. Moreover, editing of C to U at nucleotide position 2914 of the NF1 mRNA, a presumptive site for APOBEC-1 mediated editing in tumors of patients with neurofibromatosis type 1, or at any other site between nucleotide position 2879 - 3076 of NF1 mRNA could not be found in ten different types of human carcinomas. In hepatocellular and bile duct carcinoma, in breast carcinoma, in neuroendocrine carcinoma of the pancreas, in lung adenocarcinoma, in medullary thyroid carcinoma and in neuroblastoma the absence of mRNA editing was the consequence of a lack of APOBEC-1 expression. In one of the three hepatocellular carcinomas, full-length APOBEC-1 mRNA was detected by nested RT - PCR but not by ribonuclease protection assay. This tumor contained considerable amounts of the `auxiliary' components of the apo B mRNA editing enzyme, but less than 0.25% of its apo B mRNA was edited and hyperediting of apo B or NAT1 mRNA was not found. Similar levels of full-length APOBEC-1 mRNA were found in one of the two lung adenocarcinomas that contained high amounts of `auxiliary' components, but no apo B mRNA editing activity. These low levels of full-length APOBEC-1 mRNA do not promote mRNA editing even in the presence of abundant amounts of `auxiliary' components. Editing of apo B mRNA at the natural site C6666 is an excellent functional marker for the expression of APOBEC-1. At least in carcinomas that express the `auxiliary' components, the lack of apo B mRNA editing proves the absence of active APOBEC-1 and firmly excludes its oncogenic potential.

The situation in colorectal and gastric carcinoma is more complex since these tumors express APOBEC-1 mRNA to levels that are detectable by ribonuclease protection assay but do not contain the `auxiliary' components. No editing of C6666 in the apo B mRNA or hyperediting of additional cytidine residues in the apo B or NAT1 mRNA could be found in these tumors. These observations are consistent with previous findings which demonstrated a requirement of the `auxiliary' components for natural editing and hyperediting in the apo B mRNA (Yamanaka et al., 1996, Sowden et al., 1996). Supplementation of extracts from various colorectal or gastric carcinomas with nuclear extracts from Hela-cells that contain abundant amounts of these `auxiliary' components, failed to restore apo B mRNA editing activity in vitro. This experiment indicates that in colorectal and gastric carcinomas APOBEC-1 is not sufficiently expressed to induce mRNA editing, even if the `auxiliary' components are abundantly present. Therefore, also in these tumors APOBEC-1 appears to be unable to exert oncogenic effects by mRNA editing.

In colorectal and gastric carcinoma, the exon 2 skipped mRNA form was prevailing over the regularly spliced full length APOBEC-1 mRNA. The ratio of exon 2 skipped to full-length APOBEC-1 mRNA was increased in these tumors compared to normal human intestine. An overexpression of the exon 2 skipped APOBEC-1 form has recently been reported in human colon cancer and the expression of this aberrant translation product was postulated to be associated with an altered growth phenotype (Lee et al., 1998). Coexpression of the exon 2 skipped and the full-length APOBEC-1 cDNA excluded that this polypeptide of 36 amino acids interferes and possibly inhibits the editing function of APOBEC-1. According to these data, this aberrant translation product has no role in mRNA editing. The alternate splicing of APOBEC-1 mRNA appears to be a peculiar form of posttranscriptional inactivation that contributes to a presumptive down-regulation of APOBEC-1 in human carcinomas. In breast carcinoma and neuroblastoma, only the exon 2 skipped form of APOBEC-1 mRNA could be detected. Whether this is indicative for a specific regulation of APOBEC-1 mRNA splicing remains to be established.

Besides the exon 2 skipped form, no other mRNA variant of APOBEC-1 could be detected in colonic or gastric carcinoma. Sequencing of 5'RACE and RT - PCR products confirmed the cDNA sequence of APOBEC-1 in colorectal and gastric carcinomas without evidence for point mutations. Thus, a gain of function for APOBEC-1 in these carcinomas by specific mutations appears to be highly unlikely. 5'RACE mapped the transcriptional start sites of APOBEC-1 mRNA in normal small intestine and colorectal and gastric carcinoma approximately 25 - 30 nucleotides upstream from ATG, similarly as reported previously (Hirano et al., 1997). In human small intestine, two minor 5'RACE products were generated which extended approximately 1.2 kb upstream. These products could only be identified by hybridization and were not visible by ethidium bromide staining. It is most likely, that these two products represent the transcriptional start sites as determined by Fujino et al. (1998). According to our data, the usage of these transcriptional start sites appears to be very minor compared to the transcriptional start site close to ATG.

In humans, only in small intestine the APOBEC-1 mRNA is expressed to levels that are clearly detectable by Northern blotting (Lau et al., 1994). A survey in several fetal, postnatal and adult human tissues demonstrated a more widespread tissue expression of apo B mRNA editing in humans (Teng et al., 1990). Notably, 50 - 91% of the apo B mRNA were edited in adult human colon, >90% in adult human stomach and approximately 40% in adult human kidney (Teng et al., 1990). Thus, even low level expression of APOBEC-1 which is not detectable by Northern blotting can promote apo B mRNA editing activity. In most human carcinomas, the loss of apo B mRNA editing is due to absent or very low expression of APOBEC-1. In colorectal and gastric carcinoma the low level expression of APOBEC-1 is further silenced by the loss of the `auxiliary' components that are required for editing. The absence of mRNA editing in human carcinomas does not appear to be accidental. The expression of APOBEC-1 and mRNA editing most probably does not provide a growth advantage for tumors. A deregulation of APOBEC-1 function with aberrant hyperediting of new mRNA targets may be detrimental for tumor growth and therefore may be selected against in human carcinomas. This investigations indicates that APOBEC-1 is not a natural oncogene. Whether the loss of apo B mRNA editing is a prerequisite or a consequence of carcinogenesis remains to be investigated. The induction of hepatocellular carcinoma in APOBEC-1 transgenic animals, however, does not directly relate to natural human pathology and cannot serve as a model for hepatocarcinogenesis in human patients.

Materials and methods

Tissue specimens

Tissue specimens of 28 patients who underwent surgical resections of primary or metastatic tumors in the Department of Surgery, University Hospital Eppendorf, Hamburg, Germany, were used for this study, including colorectal carcinoma (n=10), hepatocellular carcinoma (n=3), bile duct carcinoma (n=2), gastric adenocarcinoma (n=3), pancreatic adenocarcinoma (n=2), pancreatic neuroendocrine carcinoma (n=1), breast carcinoma (n=2), lung adenocarcinoma (n=2), medullary thyroid carcinoma (n=1), soft tissue sarcoma (n=1) and neuroblastoma (n=1). After surgical resection the tumors were immediately examined by a pathologist. Homogeneous and viable portions of the resected tumors which were not required for subsequent histological assessments or for intraoperative histological examination, but were clearly identified as carcinomatous were snap-frozen in liquid nitrogen within 10 min of excision and stored in liquid nitrogen until use. The definite histological diagnosis of each tumor was established by the Institute of Pathology, University Hospital Eppendorf. All aspects of this study were approved by the ethics committee of the medical board of Hamburg (Ethik-Kommission der Ärztekammer Hamburg).

RT - PCR of apo B mRNA and APOBEC-1 mRNA

Apo B mRNA was amplified from total RNA by RT - PCR using oligonucleotides apo B2 (CAC GGA TAT GAT AGT GCT CAT CAA GAC, apo B antisense, nt 6786 - 6760) and apo B1 (CTG ACT GCT CTC ACA AAA AAG TAT AGA, apo B sense, nt 6552 - 6578) as described (Greeve et al., 1993). Nested PCR (20 cycles, 1 min at 95°C, 1 min at 62°C, 2 min at 72°C) was performed using 1 mul of 1 : 100 diluted primary +RT - PCR or -RT-control PCR and the oligonucleotides apo B15 (TGC CAA AAT CAA CTT TAA TGA AAA AC, apo B sense, nt 6614 - 6639) and apo B16 (A ATC ATG TAA ATC ATA ACT ATC TTT AAT ATA CTG A, apo B antisense, nt 6708 - 6674). Primary and nested RT - PCR of apo B mRNA with their respective controls were analysed by electrophoresis on 1.5% agarose gels. APOBEC-1 mRNA was amplified by RT - PCR from 5 mug total RNA as described above using the oligonucleotides SK1 (AGA GAC AGA GCA CCA TGA CTT CTG AGA, APOBEC-1 sense, nt -14 in the 5'UTR to +13) and SK2 (AGC ATT CCC AGC AGG GAC TCC AGG ACA, APOBEC-1 antisense, nt 323 - 297). Nested RT - PCR of APOBEC-1 mRNA was performed with 1 mul of 1 : 100 diluted primary +RT - PCR and primary -RT-control PCR and oligonucleotides SK3 (GGT CCT TCA ACC GGT GAC CCC ACT CTG, APOBEC-1 sense, nt 16 - 42) and SK4 (ACC AGG TGA TGG AGC AGC TGA TGG ATG, APOBEC-1 antisense, nt 259 - 233) for 25 cycles (1 min at 95°C, 1 min at 64°C, 2 min at 72°C).

Analysis of apo B mRNA editing

The apo B RT - PCR products were analysed for editing by primer extension assay as described (Greeve et al., 1991, 1993). The unedited 43-nt and the edited 54-nt extension products were separated on an 8% polyacrylamid, 7 M urea sequencing gel and analysed by autoradiography and radiophosphorimaging (Greeve et al., 1996).

Analysis of aberrant hyperediting in the mRNA of apo B and of the novel APOBEC-1 target 1 (NAT1)

Apo B mRNA was amplified from two colorectal, two hepatocellular and one gastric carcinoma using oligonucleotides apo B17 (TCC AAG ATG CAG TAC TAC TTC CAC, apo B antisense, nt 6864 - 6841) and apo B1. The RT - PCR products were cloned and individual recombinant clones were analysed by primer extension or DNA sequencing (Greeve et al., 1998). The mRNA of NAT1 was amplified by RT - PCR from three colonic and two hepatocellular carcinomas using the oligonucleotides NAT-S1 (CAT TTC AGA GCT GGT GAG CAT TTC AG, NAT1 sense, nt 2260 - 2285) and NAT-S2 (CTG CAT TAC TGG CTT GAA AGA TAG T, NAT1 antisense, nt 2758 - 2734). The RT - PCR products were cloned and 12 individual recombinant NAT1 cDNA clones from each of these five tumors were sequenced.

Analysis of NF1 mRNA editing

The mRNA of NF1 was amplified from total RNA by RT - PCR with oligonucleotides NF1 (CCG AGG CAA CAG CTT GGC CAG TAA A, sense, nt 2823 - 2847) and NF2 (CTG AGG GAA ACG CTG GCT AAC CAC CTG G, antisense, nt 3132 - 3105) for 30 cycles (0.5 min at 95°C, 0.5 min at 60°C, 0.5 min at 72°C) using a Perkin Elmer 2400 thermocycler. One mul of 1 : 100 diluted primary RT - PCR was amplified by nested PCR with oligonucleotides NF3 (GAC ATT CTG TTT CAA GGT ATA TGG TGC, sense, nt 2853 - 2879) and NF4 (ATA AAC AGT GGC ACA CAC TTC GAA GTT G, antisense, nt 3103 - 3076) for 25 cycles using the conditions as described above. The RT - PCR products were analysed for editing by primer extension assay as described (Skuse et al., 1996). In addition, the RT - PCR products for NF 1 mRNA were cloned and sequenced.

Analysis of alternate splicing in the APOBEC-1 mRNA

One mul of 1 : 100 diluted primary +RT - PCR of APOBEC-1 mRNA was re-amplified by PCR using the oligonucleotides SK1 and SK4 (ACC AGG TGA TGG AGC AGC TGA TGG ATG, APOBEC-1 antisense, nt 259 - 233). These PCR-products were separated on a 2% agarose gel and stained with ethidium bromide. After transfer to nylon membranes, the blots were prehybridized for 1 h at 60°C in DIG-Easy Hyb (Boehringer, Mannheim) and hybridized for 2 h at 58°C in DIG-Easy Hyb containing 3 pmol digoxigenin (DIG)-labeled oligonucleotide SK 3. Anti-DIG-alkaline phosphatase conjugate (Boehringer, Mannheim) was applied for detection of hybridized oligonucleotides by chemiluminescence. For DIG-labeling the oligonucleotides SK3 and VW2 were 3'tailed using terminal transferase and DIG-dUTP (Boehringer, Mannheim). Quantification of the two PCR products was performed by densitometry using NIH Image version 1.55.

RNase protection assay of APOBEC-1 mRNA

APOBEC-1 mRNA was amplified from total RNA of human intestine (ClontechÒ) by RT - PCR using oligonucleotides SK4 and SK3. The PCR-product was cloned and sequenced. Radiolabeled APOBEC-1 antisense RNA was synthesized as described (Greeve et al., 1998). It contained a total of 307 bases encompassing 242 bases of the APOBEC-1 sequence (nt 259 to 17). Total RNA from normal human small intestine (20 mug, ClontechÒ) and from tumor tissues (50 mug each) that were prepared with Tri-Reagent (Molecular Research Center Inc., Cincinnati, Ohio, USA), were coprecipitated with 4´104 c.p.m. of the alpha-32P-labeled antisense RNA probe. Hybridization and RNase digestion with the Ribonuclease Protection Assay II Kit (Ambion Inc., Austin, TX, USA) and the analysis of the protected RNAs in denaturing polyacrylamide sequencing gels was performed as described (Greeve et al., 1998).

5'RACE of APOBEC-1 mRNA

Poly(A)+ RNA was prepared from total RNA of normal human small intestine (ClontechÒ) and of one colonic and one gastric carcinoma using oligo (dT) spin columns (OligotexÔ) (Qiagen, Hilden, Germany). First- and second-strand cDNA synthesis with subsequent adaptor ligation was performed with a commercially available kit (MarathonÔ cDNA amplification kit, Clontech, Paolo Alto, USA) as described (Greeve et al., 1998). PCR was performed using Advantage KlenTaq-1 Polymerase, the adaptor-specific primer AP1 and one of the following APOBEC-1 antisense oligonucleotides: SK9 (GAT GTG TGG CGG AAT CGT TTG GTA ATG G, nt 665 - 639), SK10 (TGT GGC CAG TGA GCT TCA TCC, nt 512 - 485) and SK2. Nested 5'RACE was performed with the adaptor-specific primer AP2 and the following APOBEC-1 antisense oligonucleotides: SK10, SK2 or SK4. The primary and nested 5'RACE products were separated on a 1.0% agarose gel, blotted onto nitrocellulose and hybridized with DIG-labeled oligonucleotide SK4 (primary 5'RACE) or VW2 (nested 5'RACE). The primary and nested 5'RACE products were cloned and sequenced. The 3'end of APOBEC-1 mRNA was amplified from the cDNA pool by PCR with oligonucleotides SK6 (ACA TTT CTA GAT TCT AAA TGG C, antisense, 3'UTR, nt +86 to +64) and SK5 (CGC ACT GGA GCT GCA CTG CAT, APOBEC-1 cDNA sequence, nt 533 - 553), cloned and sequenced.

Generation of mono- and bicistronic expression vectors for expression of full-length and exon 2 skipped APOBEC-1 cDNA

The exon 2 skipped APOBEC-1 form was amplified with oligonucleotides SK-BspH1 (ACT CAT GAC TTA TGA GAA AGG AGA AG, APOBEC-1 sense spanning the exon 1/exon 2 junction plus a BspH1 restriction site at the 5'end) and SK-Spe1 (GCG ACT AGT GAT GGA GCA GCT GAT GGA TG, SK4 plus an Spe1 site at the 5'end) and cloned into the NcoI-Spe sites of the plasmid pBSKS-IRES containing the internal ribosome entry sequence (IRES) of the encephalomyocarditis virus to generate pBSKS-IRES-pp36aa. The full-length human APOBEC-1 cDNA sequence was amplified by RT - PCR from human total intestinal RNA (ClontechÒ) using the oligonucleotides SK8 (SK1 plus XbaI site at the 5'end) and SK6 (ACA TTT CTA GAT TCT AAA TGG C, antisense, nt 86 - 65 of the 3' untranslated region of APOBEC-1) and cloned into the pGEM-T easy vector. The XbaI-fragment containing the full-length human APOBEC-1 cDNA was cloned into the XbaI site of the expression plasmid pSVL (Pharmacia) to generate pSVL-hAPOBEC-1. The BamHI-fragment of pBSKS-IRES-pp36aa was inserted into the BamHI site of pSVL-hAPOBEC-1 in 3'position of APOBEC-1 to generate pSVL-hAPOBEC-1-IRES-pp36aa. pSVL-pp36-aa was generated by inserting a SacI - SacII fragment from pGEM-T-easy containing the 245 bp RT - PCR of exon 2 skipped APOBEC-1 mRNA into pSVL. The control construct pSVL-beta-gal was generated by inserting a 3.2 kb XbaI-fragment of CMV-beta-gal (Promega) into the XbaI site of pSVL. For transsient transfections FuGENEÔ 6 transfection reagent (Boehringer, Mannheim) was used.

Expression of APOBEC-1 in insect Sf9 cells

The full-length cDNA of rat APOBEC-1 (Greeve et al., 1996) was cloned into the BamHI site of pBacPAK-His3 transfer vector (ClontechÒ) and cotransfected into Sf9 cells with Bsu36I digested BacPAK6 viral DNA. Recombinant baculovirus was plaque-purified and amplified on Sf9 cells grown in monolayers. For expression of APOBEC-1 protein, 2.5´108 Sf9 cells were infected with the APOBEC-1 recombinant baculovirus at a MOI of 20 for 80 h. The Sf9 cells were resuspended in 5 ml buffer A (20 mM HEPES, pH 8.0, 5 mM beta-mercaptoethanol) and homogenized with 20 strokes of a Dounce homogenizer using a tight fitting pestel. The homogenate was adjusted to 100 mM NaCl, 20% glycerol, centrifuged for 30 min at 100 000 g using a SW40 rotor in a Beckman ultracentrifuge and stored at -80°.

In vitro editing reactions using S100 extracts from human carcinomas

200 mg of tumor tissue was grinded in liquid nitrogen and resuspended in 500 mul of 20 mM HEPES, pH 7.4, 100 mM KCl, 1 mM DTT, containing leupeptin, pepstatin, aprotinin, benzamidine and phenylmethyl-sulphonyl fluoride, each at a concentration of 10 mug/ml. After centrifugation at 12 000 g for 10 min, the supernatant was centrifuged for 30 min at 100 000 g in a Beckman TS 100 centrifuge and stored at -80°C. For in vitro editing reactions, an apo B RNA transcript of 448 bases was used (Greeve et al., 1991). Twenty mug protein of the S100 extract from the tumor tissues was incubated without and with 2 mug of Sf9 cell-extract containing recombinant APOBEC-1 and analysed for in vitro editing (Greeve et al., 1991). Nuclear extracts of Hela-cells containing high amounts of the `auxiliary' components but no in vitro editing activity were obtained from PromegaÒ.

Acknowledgements

The contribution of Dr S von der Kammer during the early characterization of mRNA editing and APOBEC-1 expression in human carcinomas is gratefully acknowledged. This work was supported by Bundesministerium für Bildung und Forschung (BMBF), 01KV9509/0 (to J Greeve) and Deutsche Forschungsgemeinschaft (DFG), SFB 545, A6 (to J Greeve).

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Figures

Figure 1 Primer extension analysis for apo B mRNA editing in human carcinomas. Apo B mRNA was amplified by RT - PCR from total RNA of human carcinomas. The RT - PCR products were analysed for editing by primer extension assay. The extension products for unedited (43 nucleotides) and edited apo B cDNA (54 nucleotides) were separated on an 8% polyacrylamide, 7 M urea sequencing gel. Representative autoradiographs for the individual tumor specimens are shown. The results for positive (TAA) and negative (CAA) plasmid DNA controls and for normal human small intestine and normal human liver are shown below

Figure 2 (a) RT - PCR for NF1 mRNA in human carcinomas. NF1 mRNA was amplified by nested RT - PCR from total RNA of 11 different types of human carcinomas. The PCR reactions and their -RT-controls were separated on a 1.5% agarose and stained with ethidium bromide. (b) Primer extension analysis for NF1 mRNA editing in human carcinomas. The RT - PCR products of NF1 mRNA were analysed for editing by primer extension assay. The extension products were separated on a 8% polyacrylamide, 7 M urea sequencing gel and autoradiographed

Figure 3 Nested RT - PCR for detection of regularly and alternatively spliced APOBEC-1 mRNA in human carcinomas. APOBEC-1 mRNA was amplified by primary RT - PCR from normal human small intestine and ten different carcinomas using oligonucleotides SK1 and SK2. One mul of 1 : 100 diluted primary RT - PCR was reamplified using oligonucleotides SK1 and SK4. The PCR product were separated on a 2% agarose gel, stained with ethidium bromide, blotted onto a nylon-membrane and hybridized with oligonucleotide VW2 specific for exon 3. Subsequently, the blots were stripped and hybridized with oligonucleotide SK3 specific for exon 2. Hybridization to DIG-labeled oligonucleotides SK3 and VW2 was detected by anti-DIG-alkaline phosphatase conjugate and chemiluminescence. DIG-labeled DNA molecular weight markers were separated in the first lane of the gel

Figure 4 Ribonuclease protection assay for determination of APOBEC-1 mRNA in human carcinomas. Total RNA from normal human intestine (20 mug) and 11 tumors (50 mug each) was hybridized with 4´104 c.p.m. of alpha-32P labeled antisense RNA probe for 18 h at 42°C in 20 mul solution A of the Ribonuclease Protection Assay II Kit (Ambion Inc., Austin, TX, USA). After digestion with RNase A and RNase T1, the RNA fragments were separated on a 6% polyacrylamide, 7 M urea sequencing gel and detected by autoradiography for 120 h. The length of the antisense transcript is 307 nucleotides. Regularly spliced APOBEC-1 mRNA containing exon 2 results in a protected RNA fragment of 242 nucleotides and exon 2 skipped APOBEC-1 mRNA of 214 nucleotides. A ribonuclease protection assay with a -actin antisense RNA is shown below demonstrating equal amounts of RNA

Figure 5 5'RACE for determination of structural integrity and transcriptional start sites of APOBEC-1 mRNA in colorectal and gastric carcinoma. First- and second-strand cDNA was synthesized from poly(A)+ RNA of normal small intestine and colonic and gastric carcinoma with subsequent adaptor ligation using the MarathonÔ cDNA amplification kit (Clontech, Polo Alto, USA). For primary 5'RACE (upper panel), the gene-specific primers as indicated were used with the adaptor-specific primer AP1. For nested 5'RACE (lower panel), 1 mul of 1 : 100 diluted primary 5'RACE was reamplified with the nested gene-specific primers as indicated and the nested adaptor-specific primer AP2. The 5'RACE products were separated on a 1.0% agarose gel, blotted onto nylon membranes, hybridized with DIG-labeled oligonucleotides SK4 (primary 5'RACE) or VW2 (nested 5'RACE) and detected by anti-DIG-alkaline phosphatase conjugate and subsequent chemiluminescence. DIG-labeled DNA molecular weight markers were separated in the first lane of the gel

Figure 6 Coexpression of full-length and exon 2 skipped APOBEC-1 mRNA in human hepatoma HuH-7 cells. Left panel: Human hepatoma HuH-7 cells (3´105 cells) were transfected with 0.4 mug of pSVL-hAPOBEC-1 and increasing concentrations of pSVL-pp36aa (0 - 1.6 mug) expressing the exon 2 skipped form of APOBEC-1. pSVL-beta-Gal was added to a total amount of 2.0 mug DNA. Two days after transfection, the cellular apo B mRNA was analysed for editing by primer extension assay (left panel). Right panel: The bicistronic expression plasmid pSVL-hAPOBEC-1-IRES-pp36aa in which both the full-length and the exon 2 skipped APOBEC-1 cDNA are expressed by the use of the internal ribosome entry site (IRES) of encephalomyocarditis virus was transfected in human hepatoma HuH-7 cells. As a control, the same amount of pSVL-hAPOBEC-1 expressing only full-length human APOBEC-1 was transfected under exactly the same conditions. Two days after transfection the cellular apo B mRNA was analysed for editing by primer extension assay (right panel)

Figure 7 Complementation of S100 extracts from human carcinomas with S100 extracts of insect Sf9 cells expressing recombinant APOBEC-1. S100 extracts (20 mug protein) from ten different human carcinoma specimens were incubated for 2 h at 30°C with a synthetic apo B RNA in the absence or presence of insect Sf9 cell extracts (2 mug protein) containing recombinant APOBEC-1. As controls (left panel), the Sf9 cell extracts were incubated with the synthetic apo B RNA in the absence and presence of Hela-cell nuclear extracts (10 and 20 mug protein, PromegaÒ). The synthetic apo B RNA was analysed for editing by primer extension assay. A representative autoradiograph is shown, the positions of edited and unedited extension products are indicated

Received 3 March 1999; revised 18 June 1999; accepted 23 June 1999
4 November 1999, Volume 18, Number 46, Pages 6357-6366
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