Davide Lazzereschi

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K‐ras‐transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin‐fixed, paraffin‐embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis. Int. J. Cancer 76:806–811, 1998.© 1998 Wiley‐Liss, Inc.

A complex pattern of mutations and abnormal splicing of Smad4 is present in thyroid tumours

Sensitivity to transforming growth factor-β is impaired in thyroid tumours. Similar to Mad – Mother Against Decapentaplegic-(Smad)4 is frequently altered in cancers, but its involvement in this system is unknown. We analysed 56 thyroid tumours of various histotypes for Smad4 mutations by PCR-SSCP and sequencing, linking them to Smad4 reactivity as examined by immunohistochemistry (IHC), and 29 of them also for abnormalities in RNA expression due to alternative splicing. In all, 15/56 cases (27%), both benign and malignant lesions, harbour alterations of Smad4 coding sequence. We found several novel intragenic mutations (13 missense, two silent, one frameshift and one large insertion-deletion), with high incidence in the linker region. A subset of mutated tumours failed to express Smad4 protein by IHC. We have also detected four alternatively spliced tumour-associated Smad4 isoforms, lacking portions of the linker region, and three more due to unreported internal exon–exon rearrangements. Smad4 is both frequently mutated and deregulated by aberrant splicing in thyroid tumours and these alterations may contribute as an early event to thyroid tumorigenesis.

Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

UMAN organs for transplantation are insufficient in quantity, and for every organ transplant undertaken there is a need for an additional five to ten organs. Waiting lists are constantly growing and many patients die before an organ can be found. Researchers continue to experiment with alternatives. One of these is xenotransplantation, which uses animals as donors and which presents an entirely new set of challenges. The pig is presently considered the most likely source of organs for human xenotransplantation because it is easy to breed, has compatibly sized organs, and offers the possibility of genetic manipulation. However, organ transplantation between distantly related species, such as pigs and humans, results in hyperacute rejection (HAR), involving the complement system. It may be possible to avoid rejection reactions by genetically engineering donor animals, so that the recipient’s immune system does not act on the graft. Thus, a number of research teams, including our group, have embarked on programs to produce pigs transgenic for the human regulators of complement activation (RCAs) genes, in the attempt to produce pigs whose organs may be suitable for transplantation into humans. 1‐3 The present study reports on the production of pigs transgenic for the human regulator of complement activation human decay accelerating factor (hDAF) by spermmediated gene transfer (SMGT), a highly efficient and reproducible alternative to microinjection, presently the most widely used system for generating transgenic animals.

Restored expression of transforming growth factor β type II receptor ink-ras-transformed thyroid cells, TGFβ-resistant, reverts their malignant phenotype

Transforming growth factor β1 (TGFβ1) inhibits the growth of normal rat epithelial thyroid cells (FRTL‐5 strain) by counteracting thyrotropin (TSH)‐stimulated DNA synthesis and by slowing the cells in the G1 phase of the cell cycle. Here, we have studied two clones of FRTL‐5 thyroid cell line transformed by the wild type (wt) v‐k‐ras oncogene (K.M.A1, K.M.A2) and one clone (A6) transformed by a temperature‐sensitive (ts) v‐k‐ras mutant. Anchorage‐dependent as well as anchorage‐independent growth of these k‐ras‐transformed cells was not inhibited by TGFβ1. TGFβ1 resistance appeared to be dependent by a functional p21 k‐ras, because A6 cell growth was partially inhibited at the nonpermissive temperature (39°C). To determine the basis for TGFβ1 resistance in k‐ras‐transformed thyroid cells, we looked for possible defects in the expression of type I (TβR‐I/ALK5) and type II TGFβ receptors (TβR‐II). Lower levels of type II receptors were present in all of the k‐ras‐transformed clones, as revealed by both Northern blot and cross‐linking experiments.

Microsatellite instability in thyroid tumours and tumour- like lesions

SummaryFifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor β (TGF-β) type II receptor (TβRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with Ml at three or more loci (RER+ phenotype). No mutations in the TβRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER+), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases.

Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs.

SINCE the beginning of this century, replacement of failing human organs with their animal counterparts has been an interesting topic of debate for writers and scientists. In the 1960s, prolonged survival after kidney transplantation from chimpanzee to human was obtained in the United States and Europe. Nevertheless, both the progressive improvement in surgical technique and in immunosuppressant therapy and the availability of cadaveric organs and living donation have reduced the interest in xenotransplantation. Because of the increasing requests for organs and the lack of donors to meet that need, xenotransplantation has become a reliable option again for temporary organ replacement (eg, of heart or liver) before definitive transplant. However, primates such as chimpanzees and baboons are expensive, can carry important zoonoses, and their use is burdened by ethical implications. A better choice for xenotransplantation might be offered by swine, which are closer to humans for anatomic and metabolic features. Discordant transplantation is associated with humoral hyperacute rejection, due to preformed antibodies and complement system activation (both classical and alternative pathway). This results in massive and irreversible vascular damage and cellular necrosis. Expression of the species-specific complement activation inhibitors could prevent this kind of rejection. Organs harvested from pigs transgenic for human decay accelerating factor (hDAF or CD55), membrane cofactor protein (MCP or CD46), and CD59 could be more efficiently grafted into human recipients. Therefore, a number of research teams, including our group, have generated pigs transgenic for human negative regulators of the complement cascade. This research has been aimed to generate hDAF transgenic swine with high efficiency and reproducibility. This goal has been reached through the innovative method of sperm-mediated gene transfer (SMGT), which was developed about 10 years ago by our group. Data presented in this paper show that hDAF-positive individuals can also be used as founders of stable lines of F1 generation transgenic pigs. MATERIALS AND METHODS hDAF Transgenic F1 Offspring Generation

Heregulin-dependent autocrine loop regulates growth of K-ras but not erbB-2 transformed rat thyroid epithelial cells.

The EGF‐like family of proteins, such as epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin (AR), betacellulin (BTC), cripto‐1 (CR‐1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL‐5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL‐5H2 cell line, overexpressing erbB‐2, Kimol cells, transformed by the wild‐type K‐ras and A6 clone, transformed by a temperature sensitive K‐ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF‐like growth factors and corresponding receptors (erbB‐2, erbB‐3, and erbB‐4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL‐5 cells, both K‐ras and erbB‐2‐transformed cells expressed elevated levels of erbB‐2 receptor. Moreover, both K‐ras transformed cells, Kimol and A6 cells, but no FRTL‐5H2 cells, were found able to express also high levels of erbB‐4 receptor and HRG/NDF ligand. Treatment of K‐ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB‐2 overexpressing FRTL‐5 cells, in K‐ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB‐2/erbB‐4 receptors in an autocrine fashion. J. Cell. Physiol. 176:383–391, 1998.

hDAF expression in hearts of transgenic pigs obtained by sperm-mediated gene transfer.

TRANSPLANTATON has been the choice option to treat successfully an increasing number of acute and chronic human pathologies with declining morbidity and mortality. However, availability of organs from human donors is limited and dramatically inadequate with respect to patient requests. Xenotransplantation from large-sized mammals has thus been reconsidered as a tool to overcome the present unbalance between organ offers and requests. Pigs have been chosen because they can be easily and cheaply bred; they do not raise ethical questions—their use as alimentary resources is generally admitted; and they possess organs largely human compatible for size, anatomical organization, and physiology. Nevertheless, this option is usually compromised by the occurrence of an irreversible hyperacute vascular rejection of the animal transplanted organ as soon as it is perfused with human blood, mediated by natural preformed antibodies and by the activation of the complement cascade. Downregulating the complement system by expressing on the surface of the swine organs one or more species-specific negative modulators of the complement complexes, such as human decay accelerating factor (hDAF), could prevent the rejection of the graft. Our group has obtained a number of transgenic pigs for hDAF using sperm-mediated gene transfer (SMGT), an innovative and highly efficient and reproducible method. In SMGT, spermatozoa are employed as natural vectors of foreign DNA and are bound, captured, and internalized into recipient egg cells to generate a genetically modified progeny. Revealing the expression of the protein on the surface of the organs is an important confirmation for successful transgenesis. The aim of this report is to show, by immunohistochemical detection, the expression of hDAF in transgenic hearts, organs maximally eligible for xenotransplantation.