RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

Low absolute peripheral blood CD4+ T-cell count predicts poor prognosis in R-CHOP-treated patients with diffuse large B-cell lymphoma.

The absolute peripheral blood lymphocyte count at diagnosis is known to be a strong prognostic factor in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but it remains unclear as to which peripheral blood lymphocyte population is reflective of DLBCL prognosis. In this cohort, 355 patients with DLBCL treated with R-CHOP from 2006 to 2013 were analyzed. The low absolute CD4+ T-cell count (ACD4C) at diagnosis negatively correlated with the overall response rate and the complete response rate significantly (P<0.00001). An ACD4C<343 × 106/l had a significant negative impact on the 5-year progression-free survival and the overall survival as compared with an ACD4C⩾343 × 106/l (73.7% (95% confidence interval (CI)=66.7-79.5) versus 50.3% (95% CI=39.0-60.6), P<0.00001 and 83.3% (95% CI=77.1-88.0) versus 59.0% (95% CI=47.9-68.5), P<0.00000001, respectively). Multivariate analysis revealed that the ACD4C was an independent prognostic marker (hazard ratio=2.2 (95% CI=1.3-3.7), P<0.01). In conclusion, a low ACD4C at diagnosis served as an independent poor prognostic marker in patients with DLBCL.

Segmental copy-number variation observed in Japanese by array-CGH.

Segmental copy-number variations (CNVs) may contribute to genetic variation in humans. In this study, we examined 80 unrelated Japanese individuals using a microarray (2,238 Bac-clones) based comparative genomic hybridization (array-CGH) assay. We found a total of 251 CNVs at 30 different regions in the genome; of these, 14 (termed 'rare' CNVs) were found individually located within distinct genomic regions of 14 individuals, while the remaining 16 CNV regions (termed 'polymorphic' CNVs) were observed in two or more individuals. The rare CNVs were confirmed by quantitative polymerase chain reactions, and characterized more precisely than in previous reports using array CGH. Distinctive features of these CNVs were observed: most prominent was that the majority of the rare CNVs presented on Bac-clones that did not overlap with regions of segmental duplication. About 90% of the polymorphic CNVs observed in this population had been previously identified, with the majority of those polymorphic CNVs located in regions of segmental duplication. It is likely, therefore, that rare and polymorphic CNVs arise through different genetic mechanisms. Since more than half of the rare CNVs are novel, it is also likely that different human populations bear different CNVs, as is the case for single-nucleotide-polymorphisms (SNPs) and insertion-deletion (indel) polymorphisms.

Increased susceptibility to apoptosis in CD45(+) myeloma cells accompanied by the increased expression of VDAC1.

Expression of CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45(+) U266 proliferates in response to a growth factor, interleukin-6. Here, we show that CD45(+) myeloma cell lines were more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress, than CD45(-) cells. Reactive oxygen species and calcium ion seemed to be involved in the susceptibility to apoptosis of CD45(+) U266. The activation of the src family kinases associated with CD45 phosphatase played an important role in the augmented apoptosis in CD45(+) U266 by oxidative stress. These results indicate that the CD45-expression renders myeloma cells competent for not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45(+) myeloma cells dependently upon the circumstantial stimuli. Furthermore, voltage-dependent anion channel (VDAC) 1 was identified as a gene highly expressed in CD45(+) U266 by cDNA subtraction. The increased expression of VDAC1 seemed to augment the sensitivity to the ER-stress because the VDAC1-transfected U266 was more susceptible to the thapsigargin-induced apoptosis. Thus, CD45 expression accompanied by the increased VDAC1 expression sensitizes myeloma cells to the various extracellular stimuli that trigger apoptosis via the mitochondrial pathways.

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice.

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.

X-rays induce dose-dependent and cell cycle-independent accumulation of p21(sdi1/WAF1).

Cell cycle arrest at the G1 checkpoint is governed by a function of wild-type p53. We assessed the behavior of the sdi1 gene, which codes for a 21kDa potent inhibitor of cdk/cyclins, after X-irradiation. X-irradiation induced sdi1 mRNA accumulation and G1 arrest only in cells possessing wild-type p53. Elevation of p21(sdi1/WAF1) was preceded by p53 accumulation, which occurred despite p53 mRNA constancy in normal cells growing in the log phase. The quantity of accumulated p53 and p21(sdi1/WAF1) was radiation dose dependent. A decrease in the S phase cell population in normal cells observed after irradiation reached a minimum at less-than-maximum levels of p53 and p21(sdi1/WAF1). Furthermore, an accumulation of p53 and p21(sdi1/WAF1) was also observed when cells were synchronized in the G0, G1 and S phase and X-irradiated. These results indicated that an X-ray induced p53 and p21(sdi1/WAF1) accumulation mechanism exists throughout the cell cycle, and that the signal strength induced by X-irradiation is dose-dependent.

Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5-40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5beta, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

Telomerase activity in hybrids between telomerase-negative and telomerase-positive immortal human cells is repressed in the different complementation groups but not in the same complementation group of immortality.

The expression of telomerase is essential for cells to be immortalized, and most immortal cell lines possessed telomerase activity. Using the cell fusion technique, it has been shown that mortal and telomerase-negative phenotypes of normal cells are dominant over immortal and telomerase-positive phenotypes, suggesting that the normal cells possessed dominant repressor-type activity for telomerase expression. Several telomerase-negative immortal human cell lines were reported, in which telomerase-independent mechanisms was supposed to maintain telomere length. We aimed at seeing whether the telomerase-negative phenotype of these immortal cells is dominant over telomerase-positive phenotype of other immortal cells in correlation with cellular mortality. Results showed that, when telomerase-positive and -negative immortal parental cell lines belonging to the different complementation groups were fused, telomerase-negative mortal hybrid clones arose, i.e. telomerase-negative phenotype was dominant as well as mortal phenotype. However, when immortal hybrid cells arose from telomerase-positive and -negative immortal parents belonging to either the same or different complementation groups, they were all telomerase-positive, i.e. telomerase-negative phenotype appeared to be recessive. Telomerase-negative immortal hybrid was never established from any combinations between telomerase-negative and -positive immortal parental cells.

Simple, rapid, quantitative, and sensitive detection of telomere repeats in cell lysate by a hybridization protection assay.

BACKGROUND: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats. METHODS: We heated 5 microL of DNA solution or 10 microL of cell or tissue lysate at 95 degrees C for 5 min, mixed it with 100 microL of hybridization solution containing 3 x 10(6) relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 degrees C. We then added 300 microL of selection buffer and incubated the mixture for 10 min at 60 degrees C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube. RESULTS: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to approximately 2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required approximately 45 min from collection of cell or tissue samples. CONCLUSIONS: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.

A hVti1 homologue: its expression depends on population doubling levels in both normal and SV40-transformed human fibroblasts.

A cDNA clone was isolated by differential colony hybridization from a cDNA library prepared from life-extended SV40-transformed human fibroblasts. The clone, tentatively named N-10, was 1272 bp in length coding for 232 amino acids. Northern analysis revealed that the expression level of N-10 was increased in normal senescent and life-extended SV40-transformed fibroblasts than in their young counterparts but was not enhanced by growth arrest. The protein fused to GFP (green fluorescent protein) localized in cytoplasmic granule. Enforced expression of N-10 resulted in premature senescence in young fibroblasts. The deduced amino acid sequence of N-10 was identical to the recently reported hVti1 gene except in one amino acid: Asp24(GAC) was ours and Asn24 (AAC) was reported. Additional base differences were found, so we referred to our sequence as the hVti1 homologue. As hVti1 protein was suggested to be involved in the vesicle transport process, the homologue may be concerned with increased secretion of extracellular matrix and various cytokines associated with cellular senescence.

Gain-of-function p53 mutations enhance alteration of the T-cell receptor following X-irradiation, independently of the cell cycle and cell survival.

Missense mutations are by the far the most common types of mutations found in p53 of human tumors, suggesting that mutant p53 proteins function either by abrogating wild-type function or by gaining new oncogenic functions. To distinguish between the dominant-negative effect and gain of new function of p53 missense mutants, we measured the ability of transfected missense mutant p53s in p53-null Jurkat cells to alter T-cell receptor (TCR) surface expression. The TCR is a key signal transduction moiety common to T lymphocytes and is one of the major sites for aberrations in T-cell leukemias/lymphomas. Three p53 mutants (248trp, 249ser, and 273his) enhanced the frequency of TCR mutants after graded doses of X-radiation compared to null p53 parent- and wild-type p53-possessing normal lymphocytes; the parent Jurkat and normal lymphocyte showed no difference. These enhancements were not the results of a change in radiosensitivity or in G1 checkpoint arrest characteristics. Therefore, the creation of this mutator phenotype by missense-type p53 mutations implies that a more direct mechanism, apart from changes of cell cycle kinetics or cell death, may be responsible for the selection of certain p53 point mutations, which eventually result in the tumorigenesis of the cell.

Increase in expression levels of interferon-inducible genes in senescent human diploid fibroblasts and in SV40-transformed human fibroblasts with extended lifespan.

The normal human fibroblast line, TIG-3 which senesces at around 80 population doubling levels (PDLs), expressed interferon (IFN)-inducible genes such as 6-16, 2', 5'-oligoadenylate synthetase (2,5-A) and HLA B7 near the end of the proliferative lifespan. Other normal fibroblast line such as MRC-5 also expressed IFN-inducible genes when senesced. Clones transformed with SV40 T-antigen, which extended their proliferative lifespan by about 20-30 PDLs, also expressed IFN-inducible genes during their extended life. Anti-IFN-beta antibodies added in culture medium repressed the expression of IFN-inducible gene in both normal senescent and life-extended SV40-transformed cells. IFN-beta repressed DNA synthesis in normal TIG-3 and induced IFN-inducible genes in both normal and SV40-transformed TIG-3. Conditioned medium recovered from life-extended SV40-transformed cells contained IFN-beta, but not IFN-alpha, IFN-gamma or TNF-alpha and possessed an activity that inhibited DNA synthesis of young TIG-3. Addition of anti-IFN-beta antibodies into the medium enhanced the serum-induced DNA synthesis of near senescent (91% lifespan completed) TIG-3, while it neither induced DNA synthesis in fully senescent TIG-3 nor extended the proliferative lifespan of TIG-3. These results suggest that normal and SV40-transformed human fibroblasts increase expression of IFN-beta with increasing proliferative age especially near the end of their lifespan resulting in induction of IFN-inducible genes and possibly in growth repression.

The usefulness of severe combined immunodeficiency (SCID) mice to study human carcinogenesis.

In the present study, we engrafted normal colonic epithelial and histologically diagnosed colonic adenomas from a familial adenomatous polyposis (FAP) patient into severe combined immunodeficient (SCID) mice and subsequently examined them histologically and molecular biologically. Successful engraftment and metastasis was observed. The facts that human normal colonic epithelium and adenomatous polyps can take in SCID mice indicates the possibility that this human SCID mouse system will be useful for investigating the dynamics of human carcinogenesis in various tissues.

Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations.

New cell lines, designated 8305C and 8505C, were established from undifferentiated thyroid carcinomas of a 67 year-old-female patient and a 78-year-old-female patient, respectively. Pathologically both these primary undifferentiated carcinoma tissues contained residual well differentiated components, suggesting well differentiated to undifferentiated carcinoma progression. Cell kinetic analysis indicate that the cell population doubling time is 43 h for 8305C and 36 h for 8505C. The saturation density at confluency is 5.7 x 10(4) cells/cm2 for 8305C and 1.1 x 10(5) cells/cm2 for 8505C. To identify genetic changes that may have occurred in these two cell lines, tumor suppressor genes p53, Rb, APC and MCC were analyzed. Sequence analysis confirmed a C:G to T:A transition at the first base of p53 gene codon 273 in 8305C and a C:G to G:C transversion at the first base of p53 codon 248 in 8505C. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene from the 8505C cell line. Loss of heterozygosity of other tumor suppressor genes were not observed. Given that p53 mutations associate with undifferentiated carcinoma but not with well differentiated carcinoma during multistep carcinogenesis of the thyroid, these cell lines should prove useful for research into the role of p53 gene mutations in malignant transformation.

DNA-DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts.

We previously reported an efficient method for subtractive cDNA cloning using oligo(dT)30-Latex and polymerase chain reaction (PCR) (E. Hara et al., Nucleic Acids Res. 19, 7097-7104, 1991). The subtraction was performed by hybridization between mRNA of cell type B and the cDNA made from mRNA of cell type A using an oligo(dT)30 primer covalently linked to Latex particles in an Eppendorf tube. The mRNA common to both types of cells could be removed by a brief centrifugation. In the present paper, the method was improved by using the sense strand DNA instead of mRNA for hybridization to cDNA covalently linked to the particles to minimize mRNA degradation and by optimizing the hybridization condition. The sense strand DNA was made from cDNA-oligo(dT)30-Latex by asymmetric PCR. Using the improved method, a subtractive cDNA library with longer cDNA inserts was successfully constructed with higher probability than the original method.

In vitro irradiation is able to cause RET oncogene rearrangement.

Elevated risk of thyroid cancers among the atomic bomb survivors as compared to the nonexposed population suggests that some genetic events related to thyroid cancer must be caused by ionizing radiation. Accordingly, inducibility of RET oncogene rearrangements, i.e., the generation of the RET-PTC oncogene, specific for thyroid cancer, was investigated among human undifferentiated thyroid carcinoma cells (8505C), which do not have RET oncogene rearrangement, after 0, 10, 50, and 100 Gy of in vitro X-irradiation by means of reverse transcription polymerase chain reaction. After testing 10(8) cells at each dose point, 3 independent samples obtained with 50 Gy of X-irradiation and 6 independent samples obtained with 100 Gy of X-irradiation showed a rearranged RET oncogene amplified band. No rearranged transcripts were obtained from cells irradiated with 0 or 10 Gy. All of the transcripts were sequenced and found to contain the D10S170 and RET sequence. Interestingly, two types of rearrangements were included in these transcripts: one is specific for thyroid cancer and the other, which contains a 150-base pair insert, is atypical, not usually seen in vivo. This insert was found to be the exon of D10S170. Furthermore, in fibrosarcoma cells (HT1080), X-irradiation also induced RET oncogene rearrangements, which included the same two types of rearrangements observed in the X-irradiated thyroid cells (8505C). These results are in favor of the hypothesis that some radiation-induced thyroid cancers, including those among atomic bomb survivors, might have developed when a growth advantage was obtained through a specific form of RET oncogene rearrangement induced by radiation exposure.