Human Vascular Microphysiological System for in vitro Drug Screening.

In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 µM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 µM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

A potential tumor suppressor role for Hic1 in breast cancer through transcriptional repression of ephrin-A1.

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically inactivated in various human cancers. In this study, we show that HIC1 is a direct transcriptional repressor of the gene encoding ephrin-A1, a cell surface ligand implicated in the pathogenesis of epithelial cancers. We also show that mouse embryos lacking both Hic1 alleles manifest developmental defects spatially associated with the misexpression of ephrin-A1, and that overexpression of ephrin-A1 is a feature of tumors arising in Hic1 heterozygous mice in which the remaining wild-type allele is epigenetically silenced. In breast cancer, we find that ephrin-A1 expression is common in vivo, but that in cell culture, expression of the EphA receptors is predominant. Restoration of HIC1 function in breast cancer cells leads to a reduction in tumor growth in vivo, an effect that can be partially rescued by co-overexpression of ephrin-A1. Interestingly, overexpression of ephrin-A1 in vitro triggers downregulation of EphA2 and EphA4 levels, resulting in an expression pattern similar to that seen in vivo. We conclude that Hic1 spatially restricts ephrin-A1 expression in development, and that upregulated expression of ephrin-A1 resulting from epigenetic silencing of HIC1 in cancer cells may be an important mechanism in epithelial malignancy.

CpG methylation is maintained in human cancer cells lacking DNMT1.

Hypermethylation is associated with the silencing of tumour susceptibility genes in several forms of cancer; however, the mechanisms responsible for this aberrant methylation are poorly understood. The prototypic DNA methyltransferase, DNMT1, has been widely assumed to be responsible for most of the methylation of the human genome, including the abnormal methylation found in cancers. To test this hypothesis, we disrupted the DNMT1 gene through homologous recombination in human colorectal carcinoma cells. Here we show that cells lacking DNMT1 exhibited markedly decreased cellular DNA methyltransferase activity, but there was only a 20% decrease in overall genomic methylation. Although juxtacentromeric satellites became significantly demethylated, most of the loci that we analysed, including the tumour suppressor gene p16INK4a, remained fully methylated and silenced. These results indicate that DNMT1 has an unsuspected degree of regional specificity in human cells and that methylating activities other than DNMT1 can maintain the methylation of most of the genome.

New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase.

DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells.

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression.

An imbalance of DNA methylation, involving widespread hypomethylation, regional hypermethylation and increased cellular capacity for methylation, is characteristic of human neoplasia. This imbalance begins in preneoplastic cells and becomes more extensive throughout subsequent stages of tumor progression. In normal cells, a primary function of DNA methylation may be to modulate compartmentalization of DNA to ensure that regional areas of transcriptionally active chromatin replicate earlier than the bulk transcriptionally inactive chromatin. We argue here that the altered methylation patterns observed during tumor progression, especially regional hypermethylation, may mark--or even help to establish--abnormalities of chromatin organization. In turn, these changes in chromatin structure may, through direct transcriptional inactivation of genes, predisposition to mutations, and allelic deletions, mediate the progressive losses of gene expression associated with tumor development.

High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.

DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, we have cloned and localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold). In comparison to colon mucosa from patients without neoplasia, median levels of DNA methyltransferase transcripts are 15-fold increased in histologically normal mucosa from patients with cancers or the benign polyps that can precede cancers, 60-fold increased in the premalignant polyps, and greater than 200-fold increased in the cancers. Thus, increases in DNA methyltransferase gene expression precede development of colonic neoplasia and continue during progression of colonic neoplasms. These increases may play a role in the genetic instability of cancer and mark early events in cell transformation.

Differentiation of human variant small cell lung cancer cell lines to a classic morphology by retinoic acid.

Variant small cell lung cancer (SCLC) is distinguished from the classic histology by changes in growth characteristics and morphology, c-myc amplification, a loss of some biochemical markers, and relative chemo- and radioresistance. Three variant SCLC lines were incubated in 1 microM all-trans retinoic acid. After 8-10 days, a marked change in morphology was noted in all three lines, with tight cell aggregation and central necrosis of large floating spheroids similar to classic SCLC. The retinoid-treated cells demonstrated decreases in growth rate and cloning efficiency to levels comparable with classic SCLC cell lines. Retinoic acid incubation caused a reproducible decrease in c-myc expression in variant SCLC cells, but was not noted to increase L-dopa decarboxylase, an enzyme which biochemically distinguishes classic from variant SCLC cell lines. Retinoid exposure led to an increase in HLA and Leu-7 antigens, but a reduction of antibody binding to 3-fucosyllactosamine, a dominant SCLC glycolipid antigen. Clonogenic assays revealed that the variant cells, after incubation in retinoic acid, became more sensitive to etoposide, but more resistant to Adriamycin. We conclude that exposure of variant SCLC cells to retinoic acid can lead to a morphologic phenotype similar to classic SCLC, but with substantial differences in cell biology. ?

Potentiation of interferon induction of class I major histocompatibility complex antigen expression by human tumor necrosis factor in small cell lung cancer cell lines.

The response of class I major histocompatibility complex antigen expression to in vitro administration of interferon and tumor necrosis factor alpha (TNF-alpha) was measured using class I major histocompatibility complex-deficient small cell lung cancer cell lines. Significant induction also was observed using gamma interferon (IFN-gamma) alone, whereas TNF-alpha alone yielded only modest induction. Classic small cell lung cancer cell lines NCI-H146 and NCI-H209 best demonstrated synergistic HLA and beta 2-microglobulin antigen induction with IFN-gamma and TNF-alpha with the following dose schedule: 3-6 days of TNF-alpha (200 units/ml) followed by 48 h of IFN-gamma (100 IU/ml). Induction was quantitated using an 125I-Protein A radioimmunoassay. Synergistic induction of the HLA and beta 2-microglobulin surface antigens on NCI-H146 was also possible with alpha interferon and TNF-alpha but required a higher concentration of the interferon, i.e., 3-6 days of TNF-alpha (200 units/ml) followed by 48 h of alpha interferon (1000 units/ml). Small cell lung cancer cell line NCI-H146 was further studied for expression of major histocompatibility complex messenger RNA using the optimal doses and sequence of addition of IFN-gamma and TNF-alpha as indicated above. A significant induction with IFN-gamma alone and synergistic induction with both IFN-gamma and TNF-alpha was quantitated for both HLA-A2 and beta 2-microglobulin transcripts using Northern blot analysis. Incubation with relatively low subcytotoxic doses of IFN-gamma and TNF-alpha also resulted in a marked synergistic decrease in c-myc message.

Characterization of differentiation-inducer-resistant HL-60 cells.

Sub-lines of the cultured human promyelocytic leukemia cell line HL-60 were individually selected for their ability to sustain exponential growth in the presence of 3 structurally-unrelated inducers of granulocytic differentiation - retinoic acid (RA), dimethylsulfoxide (DMSO), and 6-thioguanine (6TG). Selections were made by step-wise augmentation to final drug concentrations of 10(-3)mM RA, 169mM (1.2%) DMSO and 0.12mM (20 micrograms ml-1) 6TG. In addition to growth resistance, cells in each sub-line displayed variable cytodifferentiation resistance to each of the 3 selective agents, which was quantitated as the ratio of the concentration of drug required to induce differentiation in 50% of the cells in each resistant sub-line versus comparably-passaged wild-type HL-60 cells. The levels of resistance/cross-resistance were as follows: RA-resistant (res) sub-line greater than 2700-fold to RA, 1.3-fold to DMSO and greater than 1.5-fold to hypoxanthine (HXN; the noncytotoxic purine base inducer analogue of 6TG); DMSO-res sub-line 2.5-fold to DMSO, 137-fold to RA and greater than 1.5-fold to HXN; and 6TG-res sub-line greater than 1.5-fold to HXN, 9-fold to RA and 1.6-fold to DMSO. These sub-lines were not cross-resistant to sodium butyrate (NaBut), a monocyte inducer, or to 12-0-tetradecanoylphorbol 13-acetate (TPA), a macrophage inducer. HL-60 sub-lines selected by exposure to a single high concentration of 5-bromo-2'-deoxyuridine (BUdR; 3.3 X 10(-2)mM) or oubain (Ou; 5 X 10(-3)mM) were not or were slightly cross-resistant to either granulocyte or monocyte inducers. Although some variations in line/sub-line phenotype were observed, this was minor compared to the quantitative variations in response to individual inducing agents. The RA-res and 6TG-res sub-lines contained numerous double minute chromosomes (indicators of amplified genes) which were either absent or present in much smaller numbers in the parental wild-type cells or in the other drug-resistant sub-lines. There was little change or a decrease in the amplification level of the known amplified oncogene c-myc in the various drug-resistant sub-lines compared to wild-type HL-60 cells. These results (a) confirm that the neutrophilic granulocytic and monocytic/macrophagic differentiation programs in HL-60 cells are mechanistically different and separable; (b) suggest that both agent-specific and common quantitative alterations contribute to the mechanism(s) for resistance to granulocyte differentiation; and (c) suggest that the latter quantitative defects could be related to amplification of genes other than c-myc.

Cytotoxic and cytodifferentiative components of 6-thioguanine resistance in HL-60 cells containing acquired double minute chromosomes.

As an experimental strategy for potentially dissociating and studying the cytotoxic and cytodifferentiative antileukemic effects of 6-thioguanine (6-TG), cultured human promyelocytic leukemia cells (HL-60) were serially selected for growth in increasing concentrations of 6-TG (0.5 to 50 micrograms/ml). Three acquired characteristics, cytotoxic resistance, cytodifferentiative resistance, and double minute chromosomes (DM), were monitored at successive 6-TG selection levels. Approximately 200-fold resistance to the cytotoxic effect of 6-TG was acquired at the first selection step, and it neither increased at higher 6-TG selection levels nor reverted to greater sensitivity in cells subcultured off of drug. This was due to the irreversible loss of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. In contrast, a lesser, not completely quantifiable, degree of resistance developed to the cytodifferentiative effects of the purine nucleobases hypoxanthine and 6-TG which varied as a function of 6-TG selection pressure. Numerous DM, not observed in the parental wild-type HL-60 cells, appeared at 6-TG (0.5 micrograms/ml) selection which varied substantially in parallel with 6-TG selection pressure up to 6-TG (20 micrograms/ml). At higher selection levels (50 micrograms/ml or prolonged culture on 20 micrograms/ml), a marked decrease in DM occurred which was associated with the acquisition of new marker chromosomes. The most consistent marker was a chromosome 6 with additional material in the short arm (6p+); this was noted as a single copy in the basal 6-TG/20 subline but as two copies (trisomy 6; 2p+) in independently selected higher 6-TG-resistant subcultures. These cytogenetic findings suggest the presence of amplified genes which increased in number and shifted from a predominance in extra-chromosomal DM to intrachromosomal sites as a function of 6-TG selection. Among the 6-TG-resistant sublines, there was no change or a decrease in the amplification level of the known amplified oncogene c-myc from that demonstrated in parental HL-60 cells. Although proof requires detailed analyses with specific gene probes, the overall results imply that: (a) the cytotoxic component of the resistance is due to an invariant loss of HPRT which, therefore, is not likely to be related to amplified genes; (b) the cytodifferentiative component of the resistance is due to a positively selectable mechanism which could be directly or indirectly related to 6-TG-selected amplified genes; and (c) variations in the cytogenetic indicators of amplified genes and the resistance to 6-TG cannot be simply ascribed to quantitative variations in c-myc amplification.

Association of globin ribonucleic acid and its precursors with the chicken erythroblast nuclear matrix.

Nuclear matrix was prepared from both erythroblasts and erythrocytes of chicken red blood cells. Greater than 90% of the globin nuclear RNA remains bound to the erythroblast nuclear matrix. There are approximately 1000 copies of globin RNA in the nucleus per cell, and most of these contain a poly(A) tail. Precursor beta globin RNA exists in four high molecular weight forms, some of which are larger than the natural beta globin gene. Most of the ribosomal RNA is lost during the preparation of an erythroblast nuclear matrix. In contrast, some of the snRNAs are specifically enriched in the erythroblast nuclear matrix. There is little or no globin nuclear RNA in the erythrocyte nuclear matrix. There appears to be no selective attachment of the globin genes to the erythroblast nuclear matrix. The nuclear matrix is postulated to be a platform for the differential processing of nuclear RNA.