Promoter CpG methylation in cancer cells contributes to the regulation of MUC4.

Mucin 4 (MUC4) is a high molecular weight transmembrane mucin that is overexpressed in many carcinomas and is a risk factor associated with a poor prognosis. In this study, we show that the DNA methylation pattern is intimately correlated with MUC4 expression in breast, lung, pancreas and colon cancer cell lines. We mapped the DNA methylation status of 94 CpG sites from -3622 to +29 using MassARRAY analysis that utilises base-specific cleavage of nucleic acids. MUC4-negative cancer cell lines and those with low MUC4 expression (eg, A427) were highly methylated near the transcriptional start site, whereas MUC4-positive cell lines (eg, NCI-H292) had low methylation levels. Moreover, 5-aza-2'-deoxycytidine and trichostatin A treatment of MUC4-negative cells or those with low MUC4 expression caused elevation of MUC4 mRNA. Our results suggest that DNA methylation in the 5' flanking region play an important role in MUC4 gene expression in carcinomas of various organs. An understanding of epigenetic changes in MUC4 may contribute to the diagnosis of carcinogenic risk and prediction of outcome in patients with cancer.

Delayed L-DOPA-induced hyperalgesia.

Previously we reported on L-DOPA's antinociceptive effect on substance P-induced nociceptive behaviors in mice [Shimizu T, Iwata S, Morioka H, Masuyama T, Fukuda T, Nomoto M. Antinociceptive mechanism of L-DOPA. Pain 2004;110;246-9.]. Since significant hyperalgesia was noted following antinociception, our study was designed to investigate the mechanism of this hyperalgesia. Nociceptive behaviors were enhanced 2 h after L-DOPA administration. L-DOPA induced hyperalgesia occurred after conversion to dopamine because co-administration of benserazide, a DOPA decarboxylase inhibitor, completely abolished the L-DOPA-induced hyperalgesia. The D2 receptor agonist, quinpirole, depressed these behaviors entirely, while the D1 antagonist, SCH23390, inhibited the enhancement of these behaviors by L-DOPA. The D2 receptor antagonist, sulpiride, which induced hyperalgesia of the substance P-induced behaviors in naive mice, did not have any effects on L-DOPA-induced hyperalgesia. Spinal cord dopamine content increased rapidly after L-DOPA administration, exhibiting levels 100 times greater than baseline, and then returned to control after 1 h. These results suggested that the dopaminergic inhibitory system for pain sensation was temporarily impaired by excess amounts of exogenous dopamine that were derived from L-DOPA and both D1 and D2 receptors were involved in L-DOPA-induced hyperalgesia.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Transcription factor Y-box binding protein 1 binds preferentially to cisplatin-modified DNA and interacts with proliferating cell nuclear antigen.

The Y-box binding protein (YB-1) binds to inverted CCAAT box sequences that are present in the promoter region of many genes. We previously showed that YB-1 is overexpressed in human cancer cell lines that are resistant to cisplatin and that the depletion of YB-1 by transfection of a vector expressing YB-1 antisense RNA increases the sensitivity of human cancer cells to cisplatin. To determine whether YB-1 can bind to cisplatin-modified DNA, we fused YB-1 cDNA to glutathione S-transferase (GST) cDNA and purified the resulting GST fusion protein. When we tested the fusion protein with unmodified or cisplatin-modified oligonucleotides, we found that GST-YB-1 bound more strongly to cisplatin-modified oligonucleotides, as did GST fusion proteins of high mobility group 1 (HMG1), HMG2, and xeroderma pigmentosum group A protein. When we assayed the ability of proliferating cell nuclear antigen (PCNA) to interact with the GST fusion proteins, we observed binding to YB-1 but not to HMG1, HMG2, or xeroderma pigmentosum group A. Subsequent experiments demonstrated that YB-1 and PCNA interact directly via the COOH-terminal region of YB-1. Using immunochemical coprecipitation methods, we observed binding of YB-1 and PCNA in vivo. These results suggest that YB-1 can function as a recognition protein for cisplatin-damaged DNA and that it may be important in DNA repair or in directing the cellular response to DNA damage.

Transcriptional activation of the human HMG1 gene in cisplatin-resistant human cancer cells.

The nonhistone chromosomal protein, high mobility group 1 (HMG1), which is ubiquitously expressed in higher eukaryotic cells, preferentially binds to cisplatin-modified DNA. The observation that HMG1 is overexpressed in cisplatin-resistant human cancer cells suggests that cisplatin resistance may be closely associated with HMG1. To decipher the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated two overlapping genomic DNA clones containing the entire human HMG1 gene. These clones, which span approximately 15 kb of contiguous DNA, include 5 kb of the 5' flanking region as well as the entire coding sequence. We sequenced 1500 bp upstream of the first exon. The segment proximal to the transcription initiation site did not contain a TATA box but did possess an activating transcription factor site, an activator protein-2 site, one CCAAT box, and two CCAAT-binding transcription factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3-10-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB cells. An in vivo footprint experiment showed several differences of dimethyl sulfate modifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showed that binding of a nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was significantly higher than the binding of the same factor from parental cells. Semiquantitative reverse transcription-PCR and Western blot analysis also showed that expression of CTF/NF-1 was 3-20-fold higher in the resistant cell line than in its parental counterpart. These findings suggest that, in cisplatin-resistant cells, the expression of HMG1 gene product is enhanced at the transcriptional level and that this probably occurs through the enhanced expression of the CCAAT binding factor, CTF/NF-1.

Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells.

The UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (Gal NAc-T3) gene, a member of the Gal NAc transferase gene family, is expressed in a tissue-specific manner. To elucidate the function of this gene, we have focused on the molecular mechanism underlying regulation of gene expression. We have cloned Gal NAc-T3 cDNA and used it to show that Gal NAc-T3 mRNA is expressed in tumor cell lines derived from secretory epithelial tissue adenocarcinomas but not in cell lines derived from bladder and epidermoid carcinomas. Using a polyclonal antibody to Gal NAc-T3, we observed protein expression in adenocarcinoma but not non-adenocarcinoma cell lines, and in breast carcinoma cells but not in normal breast tissue. We used Gal NAc-T3 cDNA to isolate three overlapping genomic clones containing the 5'-portion of the human Gal NAc-T3 gene, and we sequenced 1.6 kb around the first exon. A transient expression assay using the luciferase gene showed that promoter activity was much higher in MCF-7 cells than in KB cells. In vivo footprint experiments showed significant protection of a distal GC box, an NRF-1 site, and an AP-2 site in MCF-7 cells. A novel stem and loop structure extending from nucleotide -103 to nucleotide -165 and contiguous to these transcription factor binding sites seemed to be functional in regulating Gal NAc-T3 gene transcription, and a KMnO4 footprint experiment showed that this stem and loop structure could be formed in vivo. We also observed dimethyl sulfate hypersensitive sites in the untranslated region around nucleotide +50 in MCF-7 but not in KB cells. These findings indicate that Gal NAc-T3 gene expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure, and that this regulation is restricted to cell lines derived from epithelial gland adenocarcinomas but not cells derived from nonsecretory epithelial tissue carcinomas. In addition, our immunohistochemical results suggest that our anti-Gal NAc-T3 antibody may be useful for diagnostic purposes in the early stages of breast cancer.

Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa.

1. Sperm nuclei were isolated and purified from boar semen by a procedure involving differential solubilization of sperm tail and acellular materials by brief exposure to reducing reagent in the presence of cationic detergent, and sedimentation through 60% sucrose. The weight ratio of DNA:RNA: total protein: protamine in this preparation was 1.00: 0.02: 1.05: 0.75, and the molar ratio of phosphorus to arginine was 1.12. 2. Boar protamine was extracted with cold acid from ethanol precipitate of reduced and carboxymethylated nuclei in 6 M guanidine hydrochloride and purified by ion-exchange chromatography on CM-cellulose. The molecular weight of the protamine was estimated to be 6600 by the gel filtration method. The protamine consisted of a single amino terminus alanine and either half-cystine or arginine as carboxy terminus, and was composed of Thr, Ser3, Pro2, Ala2, Val2, Ile, His, Half-cystine9-10 and Arg26 . 3. Chymotryptic digestion gave rise to a single amino-terminal peptide, Ala-Arg-Tyr, and two carboxy-terminal peptides, Thr-Val-Ile-Arg-Cys-Arg2-Cys and Thr-Val-Ile-Arg-Cys-Arg2, which confirmed the heterogeneity of the protamine at the carboxy-terminal end.

Analysis of cis-acting regions upstream of the rat Na+/K(+)-ATPase alpha 1 subunit gene by in vivo footprinting.

By means of in vivo footprinting, we examined the putative cis-acting DNA elements located between -50 and -122 of rat Na+/K(+)-ATPase alpha 1 subunit gene ATP1A1. Proximal and distal GC box sequences and a consensus sequence for the active transcription factor (ATF) were protected for all the tissues examined (kidney, brain and liver). Putative cooperation between two binding factors on the ATF site and the proximal GC box was observed. The overall in vivo footprinting profiles of the three tissues did not exhibit any marked differences that could account for the variation in the extent of tissue-specific transcription. The alpha 1 regulatory element (ARE) found by Suzuki-Yagawa et al. does not appear to be an element responsible for tissue-specific regulation of the gene.

Structure and functional analysis of the human STAT3 gene promoter: alteration of chromatin structure as a possible mechanism for the upregulation in cisplatin-resistant cells.

STAT3 is involved in the signal transduction activated by various cytokines and growth factors. We found that the STAT3 gene is overexpressed in cisplatin-resistant cells. We isolated a genomic fragment containing the 5'-portion of the human STAT3 gene using a bubble PCR method. Using the bubble PCR product as a probe, one genomic clone was isolated. The nucleotide sequence of the first exon and the 1800 base pairs (bps) preceding it was determined. The promoter region of the human STAT3 gene is highly homologous to the corresponding region of the mouse STAT3 gene; several potential factor binding sites such as CRE/ATF, SBE, and GC boxes are also well conserved between human and mouse. A transient expression assay using the luciferase reporter gene showed that the sequence from -403 to +102 possesses maximal promoter activity, and transcription of the STAT3 gene was significantly higher in cisplatin-resistant cells than in parental cisplatin-sensitive cells. Deletion of the region between -261 and -167 resulted in significant loss of promoter activity in both parental and cisplatin-resistant cells. In vivo footprint analysis revealed several protein bindings; however, no significant differences were observed between drug-sensitive and drug-resistant cells. MNase digestion revealed that several open or active nucleosomes were only detected in cisplatin-resistant cells. These results suggest that STAT3 promoter function in a highly structured chromatin environment requires a complex interaction of several transcriptional factors.

Cellular balance of glutathione levels through the expression of gamma-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits glutathione biosynthesis and deranges reduced glutathione (GSH) metabolism in liver cells. We isolated two BSO-resistant lines, HLE/BSO2-1 and HLE/BSO2-2, from human hepatic HLE/WT cells. Cellular levels of the Pi class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCSh) mRNA levels were markedly decreased in HLE/BSO2-1 and HLE/BSO2-2 as compared with HLE/WT. The expression of a dominant-negative mutant of c-Jun inhibited the GCSh promoter activity in HLE/WT, but not in HLE/BSO2-1. Cellular levels of AP-1, however, were not decreased in either BSO-resistant cell line. Transfection of GCSh promoter of various lengths driven reporter constructs showed no sequence-specific increase in the promoter activities in HLE/BSO2-1. However, transfection of GSTP1 cDNA into HLE/BSO2-1 and HLE/BSO2-2 restored the levels of GCSh mRNA and the GCSh promoter activity to those of HLE/WT. Sequences between -315 and -241 bp of the 5' region contained an AP-1 site responsible for the enhanced GCSh promoter activity in GSTP1 transfectants of HLE/BSO2-1. In vivo footprint analysis showed a specific protection of the AP-1 site on GCSh promoter in GSTP1 transfected HLE/BSO2-1. GSH homeostasis thus appears to be maintained by an interaction between GSTP1 and GCS in human hepatic cells resistant to the GSH poison.

Curcumin ameliorates streptozotocin-induced liver damage through modulation of endoplasmic reticulum stress-mediated apoptosis in diabetic rats.

We investigated the effect of curcumin on liver injury in diabetic rats induced by streptozotocin (STZ) through modulation of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). Experimental diabetes was induced by a single intraperitoneal injection of STZ (55 mg/kg), and curcumin was given at 100 mg/kg by gavage for 56 days. We observed that curcumin improved the morphological and histopathological changes, significantly decreased hepatic ERS marker protein: glucose-regulated protein 78, and improved liver function in diabetic rats. Moreover, treatment with curcumin markedly decreased the sub-arm of the UPR signaling protein such as phospho-double-stranded RNA-dependent protein kinase-like ER kinase, CCAAT/enhancer-binding protein homologous protein, tumor necrosis factor receptor-associated factor 2, and inositol-requiring enzyme1α; and inhibited tumor necrosis factor α, interleukin 1ß, phospho-p38 mitogen-activated protein kinase, and apoptosis signal-regulating kinase 1 in liver tissues of diabetic rats. Apoptotic and anti-apoptotic signaling proteins, such as cleaved caspase-3 and B-cell lymphoma 2, were significantly increased and decreased, respectively in diabetic rats; curcumin treatment prevented all of these alterations. In summary, our results indicate that curcumin has the potential to protect the diabetic liver by modulating hepatic ERS-mediated apoptosis, and provides a novel therapeutic strategy for the diabetic liver damage.

Postural myoclonus associated with long-term administration of neuroleptics in schizophrenic patients.

Postural myoclonus associated with long-term administration of neuroleptics was demonstrated in schizophrenic patients. Sixty patients who had been taking neuroleptics for more than 3 months were investigated for myoclonus and the relationships between postural myoclonus and age, duration of illness, duration of medication, current daily dose, cumulative dose, occurrence of abnormal finger movement, parkinsonism, and tardive dyskinesia were evaluated. Twenty-three patients (38%) showed postural myoclonus when holding the hands forward with the elbow joints flexed at about 90%. Male patients showed a higher incidence of myoclonus than female patients. Patients with myoclonus had been given significantly higher doses of neuroleptics than those without myoclonus. There was a significant correlation between the occurrence of myoclonus and abnormal finger movement. Electromyographic recordings in 7 patients with prominent myoclonus revealed that arrhythmic jerks occurred in the extensor carpi radialis and posterior deltoid muscles and that the jerks on the left and right side were not synchronized. Clonazepam reduced the frequency of the myoclonic activity.

Meclofenoxate therapy in tardive dyskinesia: a preliminary report.

Tardive dyskinesia seems to occur as a result of diminished cholinergic and enhanced dopaminergic activity in the striatum. Meclofenoxate has been shown to increase cerebral cholinergic activity. To ameliorate the tardive dyskinesia, meclofenoxate was given orally, 600-1200 mg/day, for 6-12 weeks. The effects of the drug were evaluated by scoring the degree of involuntary movement. Among 11 subjects with tardive dyskinesia or dystonia, 4 improved markedly, 1 moderately, 2 slightly, and there was no improvement in 4. One patient with subacute oral dyskinesia, induced by administration of neuroleptics for 1 month, improved markedly. The possibility that meclofenoxate may be effective in dealing with dyskinesias that are induced by neuroleptics warrants further attention.

Cell-cycle regulation of the DNA topoisomerase IIalpha promoter is mediated by proximal CCAAT boxes: possible involvement of acetylation.

Expression of DNA topoisomerase (topo) IIalpha is cell-cycle-regulated, with its peak in G(2)/M and its lowest level in G(0)/G(1). In agreement with this expression pattern, we have shown that the topo IIalpha gene promoter shows cell-cycle-dependent activity, which is repressed in G(0)/G(1) and activated exclusively in G(2)/M. However, the promoter sequence reveals no canonical CDE/CHR motifs, repressor elements commonly found in promoters of late S/G(2)-activated genes. Here, we show that at least two of the three proximal inverted CCAAT boxes (ICBs) are responsible for the G(2)/M-specific activation of the topo IIalpha promoter. Using antibody supershift experiments, we identify NF-Y as the ICB-binding transcription factor. However, the expression profile and binding capacity of NF-Y were constant during the cell cycle, suggesting a more global mechanism in topo IIalpha promoter regulation. Interestingly, we find that trichostatin A (TSA), a specific histone deacetylase inhibitor, greatly enhances topo IIalpha promoter activity in an ICB-dependent manner. In addition, the effect of TSA is predominant in G(0)/G(1) and less obvious in G(2)/M. Our data, along with the recent findings that NF-Y associates in vivo with histone acetyltransferases (HATs), strongly suggest a mechanism, in which histone deacetylation plays a crucial role in the G(0)/G(1)-specific repression of the topo IIalpha promoter, and NF-Y recruits HATs to the promoter region, thereby stimulating histone acetylation and activating transcription in G(2)/M.

The rat peptidylarginine deiminase-encoding gene: structural analysis and the 5'-flanking sequence.

Genomic clones of the rat peptidylarginine deiminase (PAD)-encoding gene (PAD) were isolated, and the gene organization was analyzed by restriction mapping and nucleotide sequencing. The PAD spans more than 50 kb and contains 16 exons and 15 introns. The lengths of the introns from 0.5 kb to more than 16.5 kb. A 1.7-kb sequence in the 5'-flanking region was determined. S1 nuclease mapping revealed two putative cap sites 79 and 81 bp upstream from the N-terminal ATG codon of PAD, which had been determined by amino acid sequence analysis. This ATG was confirmed to be the translation start site, since no other ATG codon was found in the open reading frame downstream from the cap sites. The 5'-flanking sequence contains four potential SP1-binding sites, a putative Pit-1/GHF-1-binding site, four short sequences either identical or homologous to the sequences in the promoter regions of rat or human growth hormone encoding genes, as well as a sequence similar to an estrogen-responsive element. However, neither a typical TATAA box, nor CCAAT box is present. These results provide important clues for elucidating the mechanism of female-specific and/or sex cycle-dependent gene expression.

Expression and regulation of a gene encoding neural recognition molecule NB-3 of the contactin/F3 subgroup in mouse brain.

NB-3 is a neural recognition molecule which is a member of contactin/F3 subgroup in the immunoglobulin superfamily. We report here the developmental expression pattern and localization of NB-3 mRNA in mouse brain, determination of the NB-3 gene organization and identification of the promoter region. We also describe a splicing isoform of mouse NB-3. Mouse NB-3 exhibited 96% identity with rat NB-3 at the amino acid sequence level. The splicing isoform lacked the amino acid residues between 62 and 78 of the original NB-3, which constituted a part of the first immunoglobulin-like domain. The expression of NB-3 mRNA was evident after birth, reaching a maximum at the postnatal seventh day, and declined thereafter in the cerebrum, whereas the mRNA increased in the cerebellum to adulthood. In situ hybridization demonstrated that NB-3 mRNA was preferentially expressed in the accessory olfactory bulb, layers II/III and V of the cerebral cortex, piriform cortex, anterior thalamic nuclei, locus coeruleus of the pons and mesencephalic trigeminal nucleus, and in Purkinje cells of the cerebellum. The mouse NB-3 gene consisted of 23 exons spanning more than 130kb. The overall organization of the gene was similar to those of the F11, axonin-1 and TAX-1 genes of the subgroup. By reporter gene analysis with the 5'-flanking region of the gene, we found a basal promoter activity in the 1.2kb fragment upstream of the putative transcription initiation site. This study provides a basis for elucidating the biological significance of the contactin/F3 subgroup molecules.

p53 immunoreactive stain and early colorectal adenocarcinomas.

565 cases of early colorectal adenocarcinomas were used in this study to examine mechanisms of carcinogenesis. Specimens were paraffin embedded and histological sections were stained with haematoxylin-eosin and p53. Macroscopically, early colorectal adenocarcinomas could be classified into two types: protruding and depressed. The former were composed of branching glands, while the latter were composed of straight glands which opened to the surface. The p53 positive ratio was similar for protruding tumours but was higher in depressed submucosal invasive adenocarcinomas than in depressed intramucosal adenocarcinomas. These results raise the possibility of at least two pathways for colorectal carcinogenesis, adenoma-carcinoma lesions and de novo carcinoma lesions.

A selective MAOB inhibitor Ro19-6327 potentiates the effects of levodopa on parkinsonism induced by MPTP in the common marmoset.

The effects of the selective and reversible MAOB inhibitor Ro19-6327 on parkinsonism in common marmosets, induced by MPTP were studied. Combined administration of Ro19-6327 with benserazide/levodopa potentiated the effects of levodopa to reverse akinesia. In a microdialysis study, the administration of Ro19-6327 was found to minimize the increase in metabolites of dopamine (DA) following injection of levodopa. These results suggest that Ro19-6327 suppresses the metabolism of DA, resulting in increased effects of levodopa on parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Thus, Ro19-6327 should be useful in the treatment of patients with Parkinson's disease.

Treatment with a selective MAO B inhibitor prevents loss of dopamine in the nucleus accumbens of MPTP-treated common marmosets.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to common marmosets induced motor deficits, associated with a marked decrease in the uptake of [3H]dopamine into synaptosomes in the putamen and a reduction in the content of dopamine in both caudate nucleus and nucleus accumbens. Histological analysis showed a marked loss of dopamine-containing cells in the zona compacta of the substantia nigra and less loss in the ventral tegmental area. Treatment of animals with the selective monoamine oxidase (MAO) B inhibitor, MDL 72145 (0.5 or 2.0 mg/kg) prior to, during and following administration of MPTP partially protected animals from the motor abnormalities induced by administration of MPTP alone. Both doses inhibited the onset of akinesia but only the large dose of MDL 72145 prevented the occurrence of other motor abnormalities. Moreover, while MDL 72145 (2.0 mg/kg) prevented the loss in uptake of [3H]dopamine into synaptosomes of the putamen, this was only partly prevented using the smaller dose. Similarly, while MDL 72145 (2.0 mg/kg) prevented loss of dopamine in the caudate, only partial protection was afforded by the smaller dose. However, in the nucleus accumbens, both doses of MDL 72145 prevented the depletion of dopamine, occurring in this region. Histological examination of the substantia nigra showed little or no cell loss in animals receiving the larger dose of MDL 72145 but a moderate cell loss in animals receiving the smaller dose. No damage was observed in the ventral tegmental area of animals treated with MDL 72145 (2.0 mg/kg) and only mild cell loss in those receiving the smaller dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of anticomplement agent K76 COOH on hamster-to-rat and guinea pig-to-rat heart xenotransplantation.

In normal rats, the xenobiotic K76 inhibited the C5 and probably the C2 and C3 steps of complement and effectively depressed classical complement pathway activity, alternative complement pathway activity, and the C3 complement component during and well beyond the drug's 3-hr half-life. It was tested alone and with intramuscular tacrolimus (TAC) and/or intragastric cyclophosphamide (CP) in rat recipients of heterotopic hearts from guinea pig (discordant) and hamster (concordant) donors. Single prevascularization doses of 100 and 200 mg/kg increased the median survival time of guinea pig hearts from 0.17 hr in untreated controls to 1.7 hr and 10.2 hr, respectively; with repeated injections of the 200-mg dose every 9-12 hr, graft survival time was increased to 18.1 hr. Pretreatment of guinea pig heart recipients for 10 days with TAC and CP, with or without perioperative splenectomy or infusion of donor bone marrow, further increased median graft survival time to 24 hr. Among the guinea pig recipients, the majority of treated animals died with a beating heart from respiratory failure that was ascribed to anaphylatoxins. Hamster heart survival also was increased with monotherapy using 200 mg/kg b.i.d. i.v. K76 (limited by protocol to 6 days), but only from 3 to 4 days. Survival was prolonged to 7 days with the addition of K76 of intragastric CP at 5 mg/kg per day begun 1 day before operation (to a limit of 9 days); it was prolonged to 4.5 days with the addition of intramuscular TAC at 2 mg/kg per day beginning on the day of transplantation and continued indefinitely. In contrast to the limited efficacy of the single drugs, or any two drugs in combination, the three drugs together (K76, CP, and TAC) in the same dose schedules increased median graft survival time to 61 days. Antihamster antibodies rapidly increased during the first 5 days after transplantation, and plateaued at an abnormal level in animals with long graft survival times without immediate humoral rejection. However, rejection could not be reliably prevented, and was present even in most of the xenografts recovered from most of the animals dying (usually from infection) with a beating heart. Thus, although effective complement inhibition with K76 was achieved in both guinea pig- and hamster-to-rat heart transplant models, the results suggest that effective interruption of the complement cascade will have a limited role, if any, in the induction of xenograft acceptance.