Risk factors increasing blood pressure in Japanese colorectal cancer patients treated with bevacizumab.

Bevacizumab has been reported to increase blood pressure. However, the factors, including patient characteristics and laboratory data contributing to this side effect remain unclear. Therefore, we investigated the relationships between increased blood pressure and bevacizumab administration, patient characteristics, and laboratory data. Between April 2007 and January 2018, factor analysis was retrospectively conducted by monitoring increases in blood pressure, the status of bevacizumab administration, patient characteristics, and laboratory data before the first administration in Japanese patients with colorectal cancer who satisfied the criteria for this study. Sixty-seven patients were included, 34 of whom (50.7%) had an increase in blood pressure after bevacizumab administration. On univariate analysis, liver metastasis, antihypertensive drug use, systolic blood pressure at rest before the first bevacizumab administration, body mass index, creatinine, and blood platelet count were significantly different between the two groups. Multivariate analysis was conducted using increased blood pressure as an objective variable and the factors extracted by the univariate analysis as explanatory variables. The results suggested that liver metastasis, antihypertensive drugs, systolic blood pressure at rest before the first bevacizumab administration, and creatinine were associated with the increase in blood pressure. Furthermore, a log-rank test performed based on Kaplan-Meier curves demonstrated that liver metastasis in patients not taking antihypertensive drugs and antihypertensive drug use in patients without liver metastasis were significantly associated with increased blood pressure. Additionally, liver metastasis in patients with antihypertensive drug use was significantly associated with increased blood pressure. Our findings suggest that liver metastasis and antihypertensive drug use, which was previously reported, are risk factors for increased blood pressure.

Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis.

The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.

Coexpression of translocated and normal c-myc oncogenes in hybrids between Daudi and lymphoblastoid cells.

Mechanisms that affect the transcription of the c-myc oncogene take part in the development of B-cell neoplasias such as Burkitt's lymphoma. Daudi Burkitt lymphoma cells, which express only the translocated c-myc oncogene, were hybridized with human lymphoblastoid cells, which express the normal c-myc gene; the hybrids were phenotypically lymphoblastoid and expressed both the translocated and the normal c-myc gene. This result contrasts with the findings that the decapitated c-myc gene, translocated to an immunoglobulin switch mu or alpha region, is transcriptionally silent in lymphoblastoid hybrids. Thus, there may be at least two distinct enhancer-like elements capable of deregulating c-myc transcription in lymphomas and leukemias with t(8;14) chromosome translocations. In addition, since the Daudi X lymphoblastoid hybrids express both the translocated and the normal c-myc gene, the c-myc gene product does not autoregulate c-myc transcription.

Repression of rearranged mu gene and translocated c-myc in mouse 3T3 cells X Burkitt lymphoma cell hybrids.

The productively rearranged immunoglobulin mu chain gene and the translocated cellular oncogene c-myc are transcribed at high levels both in human Burkitt lymphoma cells carrying the t(8;14) chromosome translocation and in mouse plasmacytoma X Burkitt lymphoma cell hybrids. In the experiments reported here these genes were found to be repressed in mouse 3T3 fibroblast X Burkitt lymphoma cell hybrids. Such repression probably occurs at the transcriptional level since no human mu- and c-myc messenger RNA's are detectable in hybrid clones carrying the corresponding genes. It is therefore concluded that the ability to express these genes requires a differential B cell environment. The results suggest that the 3T3 cell assay may not be suitable to detect oncogenes directly involved in human B cell oncogenesis, since 3T3 cells apparently are incapable of transcribing an oncogene that is highly active in malignant B cells with specific chromosomal translocations.

Differential expression of the translocated and the untranslocated c-myc oncogene in Burkitt lymphoma.

Burkitt lymphoma cells carrying either a rearranged or unrearranged c-myc oncogene were examined with the use of probes from the 5' exon and for the second and third exon of the oncogene. The results indicate that the normal c-myc gene on chromosome 8 and the 5' noncoding and 3' coding segments of the c-myc oncogene separated by the chromosomal translocation are under different transcriptional control in the lymphoma cells. Burkitt lymphoma cells carrying a translocated but unrearranged c-myc oncogene express normal c-myc transcripts. In contrast, lymphoma cells carrying a c-myc gene rearranged head to head with the immunoglobulin constant mu region gene express c-myc transcripts lacking the normal untranslated leader.

Deregulation of c-myc by translocation of the alpha-locus of the T-cell receptor in T-cell leukemias.

Two human T-cell leukemias carrying a t(8;14)(q24;q11) chromosome translocation were studied for rearrangements and expression of the c-myc oncogene. For one leukemia, rearrangement was detected in a region immediately distal (3') to the c-myc locus; no rearrangements of c-myc were observed in the second case (DeF). However, studies with hybrids between human and mouse leukemic T cells indicated that in the leukemic cells of DeF, the breakpoint in chromosome 14 occurred between genes for the variable (V alpha) and the constant (C alpha) regions for the alpha chain of the T-cell receptor. The C alpha locus had translocated to a region more than 38 kilobases 3' to the involved c-myc oncogene. Since human c-myc transcripts were expressed only in hybrids carrying the 8q+ chromosome but not in hybrids containing the normal chromosome 8, it is concluded that the translocation of the C alpha locus 3' to the c-myc oncogene can result in its transcriptional deregulation.

Sequences involved in accurate and efficient transcription of human c-myc genes microinjected into frog oocytes.

By microinjecting a series of deletion mutant constructs into Xenopus laevis oocytes, transcriptional control regions, two promoters, of the human c-myc gene were defined. In the case of the first promoter, sequences between -60 and -37 relative to the transcription start site contained an element essential for promoter activity. In the case of the second promoter, sequences between -66 and -56 relative to the initiation site appeared to be involved in accurate and efficient transcription. In both cases, the region identified as the essential promoter element contained GGGCGG or GGCGGG,GC box-like sequences, suggesting that c-myc gene promoter activity may be controlled by transcription factor Sp1 binding in the microinjected oocytes.

Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells.

Mouse 3T3 cells were transformed with an antisense c-fos gene fused to a mouse mammary tumor virus promoter. In transformants that integrated a large number of antisense c-fos sequences, the usual large increase in c-fos mRNA and protein following stimulation of quiescent cells by platelet-derived growth factor was blocked in the presence of dexamethasone. These cells subsequently also failed to show the stimulation of DNA synthesis normally induced by platelet-derived growth factor. Appropriate expression of c-fos appears to be a prerequisite for reentry of quiescent cells into the cell cycle.

Cell cycle expression of RNA duplex unwindase activity in mammalian cells.

An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.

Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.

Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases.

Double-stranded (ds) RNA-specific adenosine deaminase converts adenosine residues into inosines in dsRNA and edits transcripts of certain cellular and viral genes such as glutamate receptor (GluR) subunits and hepatitis delta antigen. The first member of this type of deaminase, DRADA1, has been recently cloned based on the amino acid sequence information derived from biochemically purified proteins. Our search for DRADA1-like genes through expressed sequence tag databases led to the cloning of the second member of this class of enzyme, DRADA2, which has a high degree of sequence homology to DRADA1 yet exhibits a distinctive RNA editing site selectivity. There are four differentially spliced isoforms of human DRADA2. These different isoforms of recombinant DRADA2 proteins, including one which is a human homolog of the recently reported rat RED1, were analyzed in vitro for their GluR B subunit (GluR-B) RNA editing site selectivity. As originally reported for rat RED1, the DRADA2a and -2b isoforms edit GluR-B RNA efficiently at the so-called Q/R site, whereas DRADA1 barely edits this site. In contrast, the R/G site of GluR-B RNA was edited efficiently by the DRADA2a and -2b isoforms as well as DRADA1. Isoforms DRADA2c and -2d, which have a distinctive truncated shorter C-terminal structure, displayed weak adenosine-to-inosine conversion activity but no editing activity tested at three known sites of GluR-B RNA. The possible role of these DRADA2c and -2d isoforms in the regulatory mechanism of RNA editing is discussed.

Cloning and sequencing of a c-myc oncogene in a Burkitt's lymphoma cell line that is translocated to a germ line alpha switch region.

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.

The mechanism of inactivation of the normal c-myc gene locus in human Burkitt lymphoma cells.

In human Burkitt lymphoma, the expression of the c-myc allele that has been translocated to the immunoglobulin gene locus is constitutively elevated, whereas transcription from the normal untranslocated allele on chromosome 8 is usually undetectable. Our experiments were carried out to check several plausible mechanisms proposed to explain this selective inactivation. By examining the c-myc transcripts in the presence of cycloheximide and/or actinomycin D and using differential S1 mapping, which distinguishes the c-myc transcripts of the translocated from the untranslocated allele, we found that the selective inactivation of the normal c-myc gene locus is not by a post-transcriptional regulatory mechanism, by autorepression from the constitutive expression of c-myc protein derived from the translocated locus, nor by suppression due to the previously proposed labile protein repressor. Our results are accommodated most easily by an inactivation mechanism that stably modifies the structure of the untranslocated c-myc gene.

Differentiation of F9 cells is independent of c-myc expression.

The level of c-myc expression rapidly decreases during in vitro induced differentiation of mouse F9 embryonic teratocarcinoma to endoderm cells, raising the question of whether down regulation of c-myc expression is a part of the mechanism regulating differentiation. We have investigated the effect of enforced increases or decreases in c-myc RNA expression in F9 cells on growth and differentiation. The enforced expression of c-myc RNA in clones transfected with sense c-myc did not inhibit their terminal differentiation. Dramatically decreased c-myc RNA expression in antisense c-myc transfected clones also did not substantially alter the differentiation pathway, although the transformed cells withdraw from the cell cycle slightly earlier than control cells during the differentiation induction. These results suggest that the mechanism controlling differentiation operates independently of the level of c-myc RNA expression in F9 teratocarcinoma cells.

Activation of cryptic promoters of human c-myc genes in microinjected Xenopus laevis oocytes.

The human cellular oncogene c-myc contains two promoters at the 5' end of its first non-coding exon. Cryptic promoters located within the first intron are also activated to synthesize aberrant c-myc mRNAs in some Burkitt lymphomas with a t(8: 14) chromosome translocation in which a part of the gene structure, often the 5' non-coding exon, is truncated. We have shown elsewhere that microinjected plasmid DNA carrying a normal, intact human c-myc gene directs efficient faithful transcription from its own two promoters in Xenopus laevis oocytes. Here, I have investigated the expression of different recombinants carrying various constructs of the c-myc gene in frog oocytes in order to understand the activation mechanism of those cryptic promoters. Aberrant c-myc transcripts initiating from cryptic promoters within the first intron are undetectable when the intact or truncated c-myc gene construct is used. However, the cryptic promoters can be activated in Xenopus oocytes if the truncated c-myc gene construct is fused with simian virus 40 sequences containing a 21 base-pair repeat and the replication origin. Xenopus oocytes will be useful for further investigation of enhancing elements involved in the translocated and activated c-myc genes in Burkitt lymphoma cells.

Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.

The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.

No effect of fluvastatin on the bone mineral density of children with minimal change glomerulonephritis and some focal mesangial cell proliferation, other than an ameliorating effect on their proteinuria.

AIM: There are conflicting data regarding the clinical benefit of the effect of HMG-CoA reductase inhibitors (statins) in osteoporosis. We have reported that fluvastatin (a statin) is effective in improving proteinuria and renal function in childhood IgA nephropathy with mild histological findings and moderate proteinuria. The aim of the present study was to clarify the effect of fluvastatin on the bone mineral density, bone metabolic markers, proteinuria, and renal function of children with minimal change glomerulonephritis with some focal mesangial cell proliferation whose glomeruli did not stain positive for IgA and on moderate proteinuria. PATIENTS AND METHODS: We conducted a prospective controlled study of 36 children who had recently been diagnosed with normocholesterolemic minimal change glomerulonephritis with some focal mesangial cell proliferation and moderate proteinuria, and in whom strenuous exercise was restricted. The 36 patients were randomly assigned to receive 20 mg of fluvastatin (group 1) or 5 mg/kg of dipyridamole (group 2) for two years. RESULTS: By the end of the trial, there was no difference in BMD between the groups, and there were no changes in the four bone metabolic parameters. However, the urinary protein, hematuria and BUN levels had significantly decreased in group 1 compared to baseline, and the serum total protein and albumin levels and creatinine clearance had significantly increased in group 1 compared to baseline and group 2. CONCLUSIONS: The results of this study suggest that fluvastatin therapy has an antiproteinuric effect and improves renal function in moderately proteinuric patients with mild histological glomerulonephritis, but does not increase BMD.

The effect of inositol hexaphosphate on the absorption spectra of alpha and beta chains in nitrosyl hemoglobin.

The spectral changes of nitrosyl hemoglobin on addition of inositol hexaphosphate were studied in hybrid-heme hemoglobins. The results showed that the decrease in absorption in the Soret region was mainly due to a spectral change in alpha chains, and that the tension on heme in the quaternary T structure was much stronger in alpha than in beta chains.

Modulation of double-stranded RNAs in vivo by RNA duplex unwindase.

The double-stranded RNA (dsRNA) unwinding/modifying activity is a recently discovered cellular activity capable of unwinding or denaturing dsRNAs by modifying multiple adenosine residues to inosines and creating I-U mismatched base-pairings. The biological functions of this activity, which can potentially mutate the coding capacity of messenger RNAs (mRNAs), are presently not known. However, this unwinding/modifying activity is likely to affect the secondary structures, processing, and turn-over of various eukaryotic as well as viral transcripts. Although the activity was originally found and proposed as a cellular factor that interfered with the use of antisense RNA, it now appears more likely that the activity in fact may participate in antisense RNA suppression of target genes, either by altering the coding capacity of the sense mRNAs or by accelerating the degradation of duplex RNAs. Further understanding of this novel enzymatic activity, and thus, in turn, of the metabolism of dsRNAs in vivo, should allow us to derive a better strategy for designing antisense RNA.

Bone growth oscillation: longitudinal metabolic process of bone growth in congenital adrenal hyperplasia and nonendocrine short stature.

To clarify the longitudinal metabolic process of bone growth in children, we observed the relationship between the level of serum osteocalcin (OC), a marker of bone metabolism, and growth velocity in 10 prepubertal patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and 9 prepubertal patients with nonendocrine short stature (NESS), but no major hormonal abnormalities influencing bone metabolism. Observations were made every 6 months over a 7-year period. In patients with CAH who exhibited a wide variation in growth velocity during the course of the investigation, the levels of OC fluctuated over a wide range, suggesting metabolically variable bone growth. In contrast, in patients with NESS who exhibited a relatively stable growth velocity, the OC level remained within a narrow range, suggesting metabolically stable bone growth. The meaning of such divergent metabolic processes of bone growth observed in CAH and NESS and its relationship to actual bone structure or bone intensity should be further investigated.