Receptor-mediated delivery of an antisense gene to human brain cancer cells

Background

The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High‐grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR‐dependent gliomas.

Methods

A human EGFR antisense gene driven by the SV40 promoter in a non‐viral plasmid carrying elements that facilitate extra‐chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83‐14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR).

Results

Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein‐labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on β‐galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [³H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR.

Conclusion

The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70–80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors. Copyright © 2002 John Wiley & Sons, Ltd.

     

A gene sequence expressed only in undifferentiated EC, EK cells and testes.

A clone, EC1, has been isolated from a cDNA library prepared from 4-day embryoid bodies formed by suspension culture of PSMB EC cells. This clone has been used to screen a variety of RNA sources including adult tissues, embryonal carcinoma (EC), and endoderm cell lines. A 3-kb poly(A)+ RNA species was found to be present only in undifferentiated EC cells and adult mouse testes. This species was significantly reduced in testes of W/Wv mice compared with wild-type at this locus. Germ cells and their progeny are therefore implicated as the source of the RNA in testes. Hybrid-selected RNA from PSMB could be translated in vitro into a 35-kd protein, but no translatable message was evident in either PYS-2 (parietal), or PSA5-E (visceral) endoderm cell lines. DNA sequencing of the EC1 insert revealed that it is 744 bp in length, the 3' 460 bp of which are in open reading frame. Comparison with known sequences have shown no significant homology. EC1 subclones in M13 have been used to generate single-stranded probes for hybridisation to RNA in situ in tissue sections. Hybridisation of the strand complementary to RNA produces a signal limited to the central regions of embryoid bodies formed on suspension culture of embryo-derived EK cells coinciding with the presence of undifferentiated cells. Probing of a mouse genomic library and Southern blots of liver DNA with EC1 reveals that the gene is present as a single copy.     

A novel splice variant of the human dicer gene is expressed in neuroblastoma cells

Dicer is a ribonuclease playing a key role in the biogenesis of microRNAs and small interfering RNAs. Here we report the identification of a novel splice variant of human dicer gene, the first one bearing a modified coding sequence. It encodes a truncated protein, t-Dicer that lacks the dsRNA-binding domain and is defective in one of the two RNase III catalytic centers. The splice variant was found in neuroblastoma cells and in cells induced to neuronal differentiation, whereas it was not detectable in other cell lines or in normal tissues. Interestingly, it occurred in primary neuroblastic tumors, mainly in stroma poor neuroblastomas.     

rHox: A homeobox gene expressed in osteoblastic cells

Homeodomain proteins are characterized by a conserved domain with a helix-turn-helix motif. These proteins act as regulatory factors in tissue differentiation and proliferation. However, their role in the regulation of osteoblast differentiation is unknown. In this study we have identified and characterized a homeobox gene in osteoblast-like cells. This gene, termed rHox, was isolated from a cDNA library derived from rat osteoblast-like cells. The nucleotide sequence of the 1,375 base pair (bp) cDNA contains a noncoding leader sequence of 329 bp, a 735 bp open reading frame, and 312 bp of 3′ noncoding sequence. Sequence comparison demonstrates that rHox is identical to the mouse Pmx gene (also called MHox) at the amino acid level and 90% homologous at the nucleotide level. Both Southwestern blotting and gel shift analyses indicate that rHox has potential to bind both the collagen l α 1 and the osteocalcin promoters. Transfection experiments using an rHox expression vector showed a strong repression of target promoter activity, regardless of whether the target promoters contained homeodomain binding reponse elements. These data suggest that rHox is a potent negative regulator of gene expression, although the specific role of rHox in bone gene regulation remains to be determined. © 1995 Wiley-Liss, Inc.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

A single human keratin 18 gene is expressed in diverse epithelial cells of transgenic mice

The expression of keratin 18 (K18) is restricted in humans primarily to a variety of single layered or simple epithelia. However, direct introduction of a cloned K18 gene into cultured, somatic cells by DNA transfection has been shown to result in the promiscuous expression of K18 even while the endogenous mouse form of K18 (Endo B) remains silent. To determine if the cloned K18 genomic DNA fragment contains sufficient information to be regulated appropriately when subjected to a normal developmental environment, and to determine if the cloned gene is expressed in diverse epithelia, the K18 gene, including 2.5 kb of 5' flanking sequence and 3.5 kb of 3' flanking sequence, has been introduced into the germ line of mice. Mice from all three resulting K18 transgenic lines express the gene in an appropriate tissue-specific pattern that includes hepatocytes, simple epithelia of the intestinal tract, ductal cells of several glands and epithelial cells of the thymus. No expression of K18 was found in muscle, heart, or in most of the brain even in mice carrying 18 copies of the K18 gene. In most tissues, the level of K18 RNA was directly proportional to copy number and was as efficiently expressed as the endogenous Endo B gene. The K18 protein was identified by both protein blotting methods and indirect immunofluorescence staining. No pathological consequences of overexpression of the K18 gene were observed. The cloned K18 gene appears to contain all cis-acting DNA sequences necessary for appropriate expression. In addition, diverse epithelial cell types are able to express this single human gene.     

Genetic ablation of a mouse gene expressed specifically in brain.

The 1B1075 gene was initially identified from a cDNA clone of a rat brain messenger RNA expressed in particular subsets of CNS neurons and pituitary cells. Although the protein encoded by this gene is of unknown function, its sequence suggests that it may be related to secretogranin proteins, which are found in association with secretory granules in a variety of peptidergic endocrine and neuronal cells. Here we show that the mouse 1B1075 gene is located between the dilute (d) and short ear (se) genes on chromosome 9. Many different deletion mutations have previously been isolated in the genetic region that includes these genes. By producing mice carrying two deletions that overlap at the 1B1075 locus, the gene for this brain-specific message can be completely eliminated from otherwise viable animals. The animals missing the 1B1075 gene provide an important new tool for determining the function of this gene in the brain. In addition, these results provide a new molecular entry point for detailed characterization of other genes in the d-se region.     

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.     

Pleiotrophin stimulates fibroblasts and endothelial and epithelial cells and is expressed in human cancer.

Previously we reported the purification of the heparin-binding growth factor pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent growth of SW-13 cells and of normal rat kidney fibroblasts. Furthermore, SW-13 cells transfected with PTN acquired autonomous growth in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in 16 of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor growth and angiogenesis factor in addition to its role during embryonic development.     

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.     

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.     

DSCAM, a highly conserved gene in mammals, expressed in differentiating mouse brain

Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, we isolated full-length mouse and human cDNA clones, and analysed its expression during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicted at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homologue showed 90 and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an approximately 220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. Our results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain.     

A novel human gene, encoding a potential membrane protein conserved from yeast to man, is strongly expressed in testis and cancer cell lines.

We have characterized a novel human gene (C14orf1) which codes for a polypeptide homologous to the yeast protein Yer044c. Both the human and yeast proteins are predicted to be highly basic and to present several potential, evolutionarily conserved, transmembrane domains. C14orf1 mRNA was found to be particularly abundant in the adult testis and in several cancer cell lines. The gene maps to chromosome band 14q24. Further investigations should be performed to understand the role of C14orf1 in the testis and the significance of its strong expression in the cell lines studied here.     

The LGI1/epitempin gene encodes two protein isoforms differentially expressed in human brain

The leucine-rich, glioma inactivated 1 (LGI1)/Epitempin gene has been linked to two phenotypes as different as gliomagenesis and autosomal dominant lateral temporal epilepsy. Its function and the biochemical features of the encoded protein are unknown. We characterized the LGI1/Epitempin protein product by western blot analysis of mouse and human brain tissues. Two proteins of about 60 and 65 kDa were detected by an anti-LGI1 antibody within the expected molecular mass range. The two proteins appeared to reside in different subcellular compartments, as they were fractionated by differential centrifugation. The specificity of both polypeptides was validated by cell transfection assay and mass spectrometry analysis. Immunoblot analysis of protein distribution in various zones of the human brain revealed variable amounts of both proteins. Notably, these proteins were more abundant in the temporal neocortex than in the hippocampus, the difference in abundance of the 65-kDa product being particularly pronounced. These results suggest that the two protein isoforms encoded by LGI1/Epitempin are differentially expressed in the human brain, and that higher expression levels of these proteins in the lateral temporal cortex may underlie the susceptibility of this brain region to the epileptogenic effects of LGI1/Epitempin mutations.