Harald Lahm

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.

Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures.

Purpose: Knowledge about galectin expression by human tumor cells is mainly restricted to galectins-1 and -3. This study was conducted to define the gene expression pattern of all presently known human galectins in tumor cell lines of various histogenetic origin (galectinomics). Methods: The presence of mRNAs for human galectins-1, -2, -3, -4, -7, -8, and -9 was monitored by RT-PCR analyses in a panel of 61 human tumor cell lines of different origin (breast, colon, lung, brain, skin, kidney, urogenital system, hematopoietic system). Results: The validity of the technique was first confirmed by comparison of RT-PCR data with those obtained by Western blotting and cytofluorometry for galectins-1 and -3 in 18 cell lines. The following detection of a complex pattern of gene expression beyond commonly studied galectins-1 and -3 underscored the need for this fingerprinting. The most abundantly expressed message for a member of this lectin family was galectin-8 with 59 positive cell lines. With the exception of the tested lung tumors, galectin-1 and -3 transcripts were frequently expressed in the cell line panel with differences between individual cases. Positivity for galectins-2 and -4 was confined to a significant fraction of colorectal and neural tumors. Signals for galectin-9, the third known human tandem-repeat-type galectin besides -4 and -8, appeared in colorectal carcinoma cell lines with a frequency similar to that of galectin-4 but with inter-line differences. Its expression was restricted to lines of this tumor type, of the tested ovarian carcinoma, and hematopoietic malignancies. Conclusions: The results clearly demonstrate that human tumor cells express more mRNA species for galectins than those for galectins-1 and -3. To derive unequivocal diagnostic and prognostic information by immunohistochemistry on galectins with antagonistic impact on growth control and significant influence on cell adhesion, additional monitoring of these so far insufficiently studied family members is essential.

Overexpression of Insulin-Like Growth Factor-Binding Protein-2 in Transgenic Mice Reduces Postnatal Body Weight Gain

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P , 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P , 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P , 0.05) lower carcass weights of CMVIGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P , 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I. (Endocrinology 140: 5488 ‐5496, 1999)

Galectins Are Differentially Expressed in Supratentorial Pilocytic Astrocytomas, Astrocytomas, Anaplastic Astrocytomas and Glioblastomas, and Significantly Modulate Tumor Astrocyte Migration

Galectins, a family of mammalian lectins with specificity to β‐galactosides, are involved in growth‐regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer‐assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins‐1, ‐3 and‐8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins‐1, ‐3, and ‐8 genes was investigated in 8 human glioblastoma cell lines by means of RT‐PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins‐1, ‐3 and ‐8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin‐1, ‐3 and ‐8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer‐assisted phase‐contrast videomicroscopy. The data indicate that the levels of galectin‐1 and galectin‐3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin‐8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin‐3, galectin‐1, and to a lesser extent galectin‐8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins‐2, ‐4 and ‐9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Tumor galectinology: Insights into the complex network of a family of endogenous lectins

Abstractβ-Galactosides of cell surface glycoconjugates are docking sites for endogenous lectins of the galectin family. In cancer cells, primarily galectins-1 and -3 have been studied to date. With the emergence of insights into their role in growth control, resistance to or induction of apoptosis and invasive behavior the notion is supported that they can be considered as functional tumor markers. In principle, the same might hold true for the other members of the galectin family. But their expression in tumors has hitherto been a subject of attention only to a very limited extent. Pursuing our concept to define the complexity of the galectin network in cancer cells and the degree of functional overlap/divergence with diagnostic/therapeutic implications, we have introduced comprehensive RT-PCR monitoring to map their galectin gene expression. The data on so far less appreciated galectins in this context such as galectins-4 and -8 vindicate this approach. They, too, attach value to extend the immunohistochemical panel accordingly. Our initial histopathological and cell biological studies, for example on colon cancer progression, prove the merit of this procedure. Aside from the detection of gene expression profiles by RT-PCR, the detailed molecular biological monitoring yielded further important information. We describe different levels of regulation of galectin production in colon cancer cells in the cases of the tandem-repeat-type galectins-8 and -9. Isoforms for them are present with insertions into the peptide linker sequence attributed to alternative splicing. Furthermore, variants with distinct amino acid substitutions (galectin-8, Po66-CBP, PCTA-1, CocaI/II and galectin-9/ecalectin) and generation of multiple mRNA species, notably those coding for truncated galectin-8 and -9 versions with only one lectin site, justify to portray these two family members not as distinct individuals but as groups. In aggregate, the ongoing work to thoroughly chart the galectin network and to disentangle the individual functional contributions is expected to make its mark on our understanding of the malignant phenotype in certain tumor types. Published in 2004.

Interleukin-6 stimulates clonogenic growth of primary and metastatic human colon carcinoma cells

Interleukin-6 (IL-6) is a pleiotropic cytokine which exerts biological activities on various cell types including neoplastic cells. We have investigated the biological effect of IL-6 and the expression of IL-6 receptors (IL-6R) on human colorectal carcinoma cell lines. Isreco-1 was derived from the primary site of a colon cancer while Isreco-2 and Isreco-3 were established from a liver and peritoneal metastasis of the same patient. IL-6 stimulated colony formation in methylcellulose of Isreco-1 cells to 150% (P < 0.05). The effect was even more pronounced on the metastatic Isreco-2 line where colony numbers in the presence of IL-6 were enhanced up to four-fold (P < 0.0001) in a dose-dependent fashion. An anti-IL-6 antibody completely abolished this growth stimulatory effect of IL-6. RT-PCR analysis revealed transcripts for IL-6Ralpha and gp 130 in these cell lines. Experiments with additional cell lines confirmed the general expression of gp130 but showed limited expression of the IL-6Ralpha chain. Surprisingly, about half of the cell lines tested expressed IL-6 mRNA at low levels which was not translated into protein. Our results suggest that IL-6 can potently stimulate anchorage-independent growth of some colorectal carcinoma cells. This stimulation appears to occur through a paracrine mechanism.

Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins

The insulin‐like growth factor‐binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF‐independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time‐ and tissue‐specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP‐1, −2, −3, or −4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP‐1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP‐2 transgenic mice. The overexpression of IGFBP‐3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland‐targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue‐specific overexpression of IGFBP‐4 resulted in hypoplasia and reduced weight of smooth muscle‐rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice.—Schneider, M. R., Lahm, H., Wu, M., Hoeflich, A., Wolf, E. Transgenic mouse models for studying the functions of insulin‐like growth factor‐binding proteins. FASEB J. 14, 629–640 (2000)

Growth regulation and co-stimulation of human colorectal cancer cell lines by insulin-like growth factor I, II and transforming growth factor alpha.

We have tested growth factor responsiveness of a panel of eight human colorectal carcinoma cell lines. Insulin-like growth factors I and II (IGF-I and IGF-II) stimulated growth of five lines (HT-29, LS411N, LS513, SW480, WiDr). At 30 ng ml-1 both factors enhanced growth up to 3-fold. They induced half-maximal stimulation at 1.9-6.51 ng ml-1. Even after delayed addition IGF-I and II significantly enhanced growth in a short-term proliferation assay. They exerted maximal effects under limiting serum conditions (0.5% FCS) and at low cell density (1.25-5 x 10(4) ml-1). Using these conditions transforming growth factor alpha (TGF alpha) enhanced proliferation of all IGF-responsive cell lines, except SW480. 1.11-3.31 ng ml-1 were required to obtain a half-maximal response. With 10-20 ng ml-1 maximal stimulation occurred at plateau values different from those for IGF-I/II. Proliferation of all cell lines responsive to both IGF-I and TGF alpha was further enhanced by combining both factors, resulting a synergistic response of LS513, while the effects on HT-29, LS411N and WiDr were additive. With HT-29 and LS411N a 24 h exposure to TGF alpha was sufficient to obtain a full response in the co-stimulatory assay. Our results illustrate the importance of IGF-I/II and TGF alpha as stimulators of growth of colorectal carcinomas.

Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor

Background and aims: Galectins are β-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. Methods: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with α- and β-d-galactose, α-d-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. Results: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3–4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1–2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. Conclusions: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Growth Inhibition in Giant Growth Hormone Transgenic Mice by Overexpression of Insulin-Like Growth Factor-Binding Protein-2

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 μg/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 wa...