Biomedical subjects
Publications and source records attributed to S Knuutila.
We report a simple procedure that allows the application of chromosomal in situ suppression hybridization to standard G-banded and Entellan-mounted archival preparations that had been stored for 4-8 y. The procedure includes removing the cover slips in xylene, refixation in buffered formaldehyde-acetone mixture, and washing in 2 x SSC, Tween 20.
Strong endothelial proliferation is a prominent feature of glioblastomas and sometimes these proliferated areas transform into a malignant component of glioblastoma, resulting in gliosarcomas. It has not been established whether the proliferated endothelial areas are cytogenetically abnormal. To clarify this question, the most common cytogenetic aberration, gain of chromosome 7, was chosen and in situ hybridization was performed on paraffin-embedded tissue sections of three glioblastomas. The purpose was to compare the parenchymal tumor cells and the endothelial cells. The results showed trisomy 7 in only a small amount of endothelial cells (5-8%), whereas 23-38% of parenchymal tumor cells displayed trisomy 7.
Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.
The functional immunoglobulin (Ig) genes of B lymphocytes undergo somatic mutations during immune responses. These mutations modify the antigen binding site of the immunoglobulins, thereby enhancing the average affinity of the antibodies produced. The molecular mechanism underlying these B cell hypermutations remains unresolved, partly because it is difficult to grow normal B cells in long-term cell cultures and because there is no suitable transformed or malignant B cell line which generates mutations in its immunoglobulin genes in vitro. Here, we show that the recently established follicular lymphoma line HF-1.3.4 generates somatic hypermutations in vitro at a high frequency of 0.7 x 10(-6) mutations per base pair per generation in standard cell cultures (RPMI 1640 + 5% fetal calf serum). This shows for the first time that B cell hypermutation can occur without T cells or T cell factors. The mutation frequency increased approximately tenfold to 1 x 10(-5) mutations/base pair/generation with B cell-specific growth factors (interleukins-2 and -4 and three antibodies stimulatory to HF-1.3.4 cells). This HF-1.3.4 lymphoma line may help to elucidate the molecular mechanism of Ig gene hypermutation.
The cellular origin of trisomy 7 in non-neoplastic kidney tissue specimens from 10 patients, seven with malignant tumors and three with non-neoplastic kidney diseases, was studied by the MAC (morphology antibody chromosomes) technique, which allows analysis of cellular morphology/histology, immunophenotype, and chromosomal aneuploidy by conventional cytogenetics, and/or fluorescent in situ hybridization in both interphase and mitotic cells. In primary cultures, trisomy 7 was detected primarily in cytokeratin-positive cells. Among freshly isolated renal cells, the trisomy was mainly observed in proximal tubular cells positive to brush-border antigen, and, to a lesser extent, in distal tubular cells positive to Tamm-Horsfall glycoprotein. The frequency of trisomy 7 in lymphocytes expressing CD3 or CD22 antigens isolated from non-neoplastic and tumor tissues was substantially lower than in the epithelial cells and was not increased compared with that in control lymphocytes from peripheral blood. The results thus demonstrate that the non-neoplastic kidney cells with trisomy 7 are mainly normal epithelial cells, preferentially those of the proximal tubule.
We studied the origin of nucleated red blood cells (NRBC) in peripheral venous blood samples from 40 pregnant women carrying a male fetus, using a technique that allows direct chromosomal analysis by in situ hybridisation on immunologically and morphologically classified cells. Samples from ten nulligravid women were studied as controls. NRBC were enriched by negative magnetic activated cell sorting (miniMACS) using anti-CD45 monoclonal antibody. NRBC were detected by alkaline phosphatase anti-alkaline phosphatase immunostaining using a monoclonal anti-glycophorin A antibody. The origin of the NRBC was determined by fluorescence in situ hybridisation using X and Y specific probes. NRBC were found in 37 of the 40 pregnant women at a range of 1 to 230 per 20 ml of venous blood and in 6 of the 10 controls at a range of 1 to 3 per 20 ml of venous blood. All NRBC detected in the pregnant women were evidently of maternal origin, and in the pregnant women the number of NRBC was significantly higher (P < 0.05) than in the controls. Pregnancy per se seems to induce the appearance of maternal NRBC in the circulation, and it cannot therefore be assumed that NRBC isolated from the maternal blood are of fetal origin on the basis of morphology alone. Discrimination of fetal NRBC must occur for prenatal diagnosis of fetal genetic disorders.
We assessed the involvement of natural killer (NK) cells in chronic myeloid leukemia (CML). We adopted the MAC (morphology antibody chromosomes) method, which allows simultaneous assessment of cell morphology, immunophenotype, and chromosome aberrations in the same mitotic or interphase cells. We examined three patients with CML in chronic phase and two patients with the disease in blast crisis. Patients in the chronic phase of the disease showed no involvement of NK cells, but involvement was detected in one of the patients in blast crisis. In this patient, a proportion of the B cells and lymphoid stem cells was also neoplastic, whereas mature postthymic T cells were normal.
We report the results of cytogenetic analysis of malignant fibrous histiocytoma of soft tissue (MFH). Seven of 12 successfully cultured MFHs had complex clonal aberrations, including translocations, deletions, and unidentifiable marker chromosomes. Telomeric associations were observed in five and the double minute phenomenon in four of seven MFHs with abnormal karyotypes. In one case (a storiform-pleomorphic MFH, grade IV) with a complex polyploid karyotype, two clonal ring chromosomes were present, one interpreted as r(19)(p13q13), one unidentified. In two tumors, clonal structural rearrangements of chromosome 1 were seen: del(1)(q21) in a storiform-pleomorphic MFH, grade IV, and add (1)(q21 or q32), t(1;10)(p22;q22) in a myxoid MFH, grade I. The remaining five MFHs had normal karyotypes, but in two of them nonclonal, structural aberrations were found. The modal chromosome number in the studied MFHs varied widely, but the majority of tumors with abnormal karyotypes had polyploid chromosome complements (five of seven cases). Our results confirm many of the previous findings and indicate that double minutes (dmins) may be more frequent in MFH than previously reported.
Growth requirements of human natural killer cells in IL-2-supplemented cultures were studied. NK cell proliferation was monitored by the MAC (morphology antibody chromosomes) technique and subset specific cell cycle analysis, which both enable direct determination of cell growth in specific lymphocyte subsets among heterogeneous lymphocyte populations. Our results show that even in the presence of saturating concentrations of IL-2, the proliferative capacity of purified CD16+ cells is quite low, but can be stimulated in a dose dependent manner by CD4+ cells. CD4+ cells could partially be replaced by IL-4 but not by various other commercially available cytokines. These results provide further evidence of the requirement of accessory stimuli in NK cell proliferation, and support the interpretation that NK cells have a direct regulatory role in specific T cell responses.
We present here a 7 year old girl with the clinical signs of Wolf-Hirschhorn syndrome (WHS). Only on high resolution banding was a deletion of 4p16.3 suspected in both the proband and the father. Further studies using simultaneous R banding and FISH, with cosmid probe pc847.351 containing the mildly repetitive fragment 847-EC, confirmed the diagnosis and showed a paternal balanced translocation t(4;8)(p16.3;q24.3).
Chronological aging of women is clearly associated with an increase in both X-chromosome loss and micronuclei formation in peripheral lymphocytes. It has been suggested that micronucleus formation is an important mechanism of chromosome loss. In the present study, fluorescence in situ hybridization was used to study micronuclei content in two age groups (women below 30 and above 50 years old). A probe for centromeric alphoid consensus sequences (SO-alpha AllCen) and a cloned X-specific centromeric probe (pXBR) were separately used to detect the presence of any chromosomes and the X chromosome, respectively. The presence of centromere-positive micronuclei was significantly higher among the older donors (51.5%) than among the younger donors (34.3%). The X chromosome was highly overrepresented in the micronuclei, the older women showing a higher proportion of X-positive micronuclei (24.0%) than the younger women (14.0%). Assuming that the rest of the centromere-positive micronuclei contained autosomes, a significant age-dependent difference was also noted for micronuclei harboring autosomes (27.5% among the older women and 20.3% among the younger women). These findings suggest that both the X chromosome and autosomes are responsible for the age-dependent increase of micronuclei in women's peripheral lymphocytes.
S-adenosylmethionine decarboxylase (AdoMet-DC) is a key enzyme in polyamine biosynthesis. The human genome contains at least two loci for the AdoMetDC gene (AMD), one of which (AMD1) has previously been mapped to chromosome 6 and the other (AMD2) to the X chromosome. The locus on chromosome 6 is the transcriptionally active gene. We now report characterization of the AMD2 locus (GenBank Accession No. U02035) on the X chromosome, which contains sequences that cross-hybridize with human AdoMetDC cDNA. This DNA lacks all of the introns present in AMD1 and has numerous mutations in the protein-coding region. Its overall nucleotide sequence identity with AdoMetDC cDNA is about 90%. AMD2 is therefore a processed pseudogene, which, because of multiple mutations, cannot be translated to an active AdoMetDC enzyme, even if it were transcribed. Chromosomal loci for human AMD sequences were determined by in situ hybridization to metaphase chromosomes, with genomic DNAs from the active gene and the pseudogene loci as probes. AMD1 was localized to chromosome region 6q21-->q22 and AMD2 to band Xq28.
Nucleated erythrocytes found in the venous blood of pregnant women have been seen as the most promising type of cell for prenatal diagnosis of fetal abnormalities because of their short life span and their scarcity in the venous blood of healthy nonpregnant women. However, this review demonstrates that a great proportion of the nucleated erythrocytes found in venous blood during pregnancy are of maternal origin and that pregnancy per se induces nucleated erythrocytes of maternal origin to appear in the peripheral blood. More efficient and specific enrichment techniques are therefore needed to obtain purified nucleated erythrocytes of fetal origin in such quantities that reliable prenatal diagnoses can be performed.
In situ hybridization was performed to study the clinical significance of trisomy 12 in fifty patients with B-cell chronic lymphocytic leukemia at various stages of disease. Trisomy 12 was detected in 12%-65% (median 53%) of the circulating neoplastic cells in seven out of 20 patients with advanced Binet stage C disease. In contrast, 22 patients with Binet stage A and eight patients with Binet stage B disease were found to be negative for trisomy 12. As occurrence of trisomy 12 was associated with the presence of B-symptoms and hepatosplenomegaly, its association with advanced disease was further considered. In addition, atypical morphology was a common finding in trisomic patients who also displayed higher serum levels of soluble CD25 than patients without trisomy at Binet stage C. No significant differences were detected in serum levels of soluble CD8 and of soluble CD23. No correlation with a lymphocyte doubling time of < 12 months, marked lymphadenopathy, or prior treatment was apparent. However, refractoriness to treatment was evident more frequently in trisomic than in non-trisomic patients (p < .05). In conclusion, trisomy 12 in B-cell chronic lymphocytic leukemia appears to occur predominantly in advanced and symptomatic disease with atypical morphology. It could indicate a high risk for treatment failure thus serving as a marker of poor prognosis in this disease.
We looked for clonal chromosomal abnormalities in myeloid cell lineages in the bone marrow aspirates from six children with acute lymphoblastic leukemia (ALL). The study was carried out using a combination of MAC (morphology, antibody, chromosomes) and in situ hybridization procedures. In patients whose leukemic cells expressed only lymphoid antigens, we found chromosomal aberrations in CD10- and CD20/22-positive lymphoid cells. Mature CD22+ and CD3+ lymphocytes did not have the chromosomal aberrations. In one patient whose leukemic cells also expressed myeloid-associated antigens, the clonal chromosome aberrations were seen not only in the CD10+ and CD19+ blasts, but also in glycophorin A-positive morphologically nonleukemic erythroblasts.
Our main aim was to evaluate whether maternal whole venous blood could be used for determination of fetal sex, when no enrichment of fetal cells was attempted and when "standard" interphase cytogenetics and PCR analysis were adopted. Altogether 39 pregnant women were studied by using ISH and 59 by using PCR. Out of the 59 pregnant women, 26 carried a male fetus and 33 a female fetus. By ISH, Y-positive cells were detected in 12 of 19 pregnancies with a male fetus and in two of the 20 pregnancies with a female fetus. The frequency of the fetal cells ranged from 1 in 639 to 1 in 100,000. By nested PCR with primers flanking a Y-specific repeat sequence, the positive band indicating a male fetus was found in one of the 26 pregnancies with a male fetus and in one of the 33 pregnancies with a female fetus. According to our results, fetal cells in maternal blood cannot be reliably used for prenatal diagnosis without enrichment of fetal cells.
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Polyamines are found in all cells but their function is not fully understood. We have studied the effect of polyamines on the passage of cells through the cell cycle using a polyamine auxotrophic mutant, CHO-P22, which has no detectable ornithine decarboxylase activity. The ability of these cells to grow without serum allows efficient polyamine depletion. A flow cytometric analysis of DNA content and bromodeoxyuridine labeling showed that without added polyamines the cells accumulated in S-phase, the rate of DNA synthesis was retarded, and the entry into mitosis was blocked. Addition of polyamines to cultures deprived of polyamines induced cells in all phases of the cell cycle to reinitiate cycling. Earlier studies have shown that cells with damaged DNA are blocked from entering into mitosis but caffeine can partly overcome this block and induce premature chromosome condensation. Polyamine-depleted CHO-P22 cells responded to caffeine in the same way as cells with damaged DNA. These results show that polyamine depletion in CHO-P22 cells primarily affects DNA synthesis. The finding that polyamine-starved cells continuously take up bromodeoxyuridine without a corresponding increase in the amount of DNA is compatible with extensive repair of erroneous and/or damaged DNA. Polyamine auxotrophic Chinese hamster ovary (CHO) cells might be useful in studies on the regulation of mitosis in mammalian cells.