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C L Richer

Publications and source records attributed to C L Richer.

At least 37 records · Page 2Linked to original sources
PubMed1990

Chromosome evolution and high-resolution analysis of leucocytes, bone marrow, and tumor cells of retinoblastoma patients.

High-resolution cytogenetics were applied to leucocytes, bone marrow, and tumors of 8 retinoblastoma (Rb) patients in search of microdeletions or subtle rearrangements and in order to determine clonal evolution. Four of 9 tumors (Rb1, Rb6.1, Rb6.2, and Rb8) showed a deletion in the characteristic region on 13q while 2 others (Rb3 and Rb4) were hemizygous for chromosome 13 in approximately one-third of the cells. Our study presents a particularly high incidence of chromosome 13 anomalies as compared to the previously published data. Furthermore, comparison of karyotypes of 3 significant cases (Rb1, Rb6, and Rb8) allows the reconstruction of the necessary steps in the evolution of retinoblastoma. It also shows the need for a double mutation in tumor development, both in hereditary and non-hereditary cases. High-resolution chromosome analysis of retinoblastoma patients provides a rare opportunity to study the succession of events necessary for tumor development.

Bone Marrow↗
PubMed1990

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study.

A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5'-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%-85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.

Adult↗
PubMed1990

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution.

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2'-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R- and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.

Adult↗
PubMed1990

Detection by electron microscopy of a small subband 13q14.11 deletion in an hereditary retinoblastoma.

High-resolution banding, specific for electron microscopy, was applied to chromosomes of synchronized blood lymphocytes obtained from a child with bilateral retinoblastoma. Ultrastructural analysis of the subbands in region q14.1, after synchronization and immunochemical banding, showed that the deletion in the abnormal chromosome 13 corresponds to subband 14.11, thus evidencing that the retinoblastoma gene is located within subband q14.11. This first application to a diagnostic problem of immunochemical banding suggests that, coupled with electron microscopy, this banding provides a higher resolution than that obtained with light microscopy and should be useful to pinpoint important localizations.

Child, Preschool↗
PubMed1990

Ultrastructural, immunohistochemical, and cytogenetic study of a malignant peripheral neuroectodermal tumor in a patient seropositive for human immunodeficiency virus.

A case of malignant peripheral neuroectodermal tumor occurring during the course of a human immunodeficiency virus (HIV) infection is reported. The patient was a male homosexual who presented with a rapidly enlarging tumor of the posterior lower thoracic wall. By light microscopic examination the tumor was a small cell tumor showing occasional structures suggestive of Homer-Wright rosettes. The strong positivity for neuron-specific enolase and the neurosecretory granules indicated the neural differentiation of the tumor. Its precise nature was shown cytogenetically by the presence of the t(11;22) translocation, which distinguished it from the classical neuroblastoma.

Adult↗
PubMed1990

Standard karyotype of the domestic horse (Equus caballus). Committee for standardized karyotype of Equus caballus. The Second International Conference for Standardization of Domestic Animal Karyotypes, INRA, Jouy-en Josas, France, 22nd-26th May 1989.

The following decisions concerning the banded karyotype of the horse (Equus caballus) were made at the second International conference for Standardization of Domestic Animal Karyotypes, held at Jouy-en Josas, France, 22nd-26th May 1989: (1) numbering of the chromosomes was modified to correspond to an arrangement into only two groups (the non-acrocentrics and the acrocentrics) within which the autosomes are placed according to length alone; (2) a more compact karyotype arrangement was adopted: chromosomes 1 to 5 on the first row, 6 to 10 on the second, 11 to 13, and, at the far right, X and Y on the third row, 14 to 19 on the fourth row, chromosomes 20 to 25 on the fifth, and 26 to 31 on the sixth row; (3) the NOR-bearing horse chromosomes were identified as numbers 1, 28 and 31.

Animals↗
PubMed1990

Neuronal and photoreceptor differentiation of retinoblastoma in culture.

This study describes an in vitro system, which favors attachment and differentiation of primary retinoblastoma cells. Indeed, tumor cells from primary retinoblastomas were successfully grown as long-term monolayer cultures, using collagen type-1 coated flasks, preincubation of tumor cells in fetal calf serum and a medium richly supplemented with serum. This reliable system allowed growth and attachment and induced differentiation towards neuronal and photoreceptor morphology. Each of the nine tumors studied showed substantial cell proliferation and differentiation. Cells with glial characteristics were observed in lower numbers. Furthermore, in five cell-lines, some cell clusters showed pigmentation characteristic of pigment epithelial cells (PE). By electron microscopy, a significant proportion of cells showed conventional neuronal differentiation; others formed typical Flexner-Wintersteiner rosettes with individual photoreceptor cells showing a more highly differentiated pattern quite similar to fleurette inner segments. Immunohistochemical studies did not reveal evidence of transition between undifferentiated retinoblastoma cells and glial cells, although foci of multipolar cells were found resembling glial elements and containing glial fibrillary acidic protein. Thus no definite sign of glial differentiation was observed. These results show that most primary retinoblastoma cells can grow in attachment culture and differentiate into at least two distinct morphological cell types. This study also offers a potentially rewarding system to analyse the factors controlling growth and differentiation of human tumor cells in vitro.

Cell Adhesion↗
PubMed1989

Synchronization of cultured retinoblastoma cells for high-resolution chromosomes showing up to 1000 bands.

A method that allows high-resolution cytogenetic analysis of retinoblastoma cells in primary culture and subpassages is described. This method is based on the addition of high concentrations of bromodeoxyuridine or thymidine to obtain chromosomes subsequently banded to show 600-1000 bands. The results are compared with the standard harvest after 24 hours or long-term culture, and with low-temperature synchronization after long-term culture. After blocking with bromodeoxyuridine or thymidine, the chromosomes are significantly longer than after cold synchronization or after the unsynchronized techniques. When they are GTG, RHG, or GBG banded, more than 40% of the mitoses are in the earlier phases with chromosomes showing more than 600 bands per haploid set. This method significantly improves the general quality of retinoblastoma tumor cell chromosomes and increases diagnostic and prognostic accuracy.

Chromosome Aberrations↗
PubMed1989

A man with isochromosome Xq Klinefelter syndrome with lack of height increase and normal androgenization.

We report on a patient with Klinefelter syndrome (KS) and the homogeneous aneuploidy 47,Xi(Xq)Y, or male trisomy Xq. He had many characteristics of classical KS: small testes, azoospermia, elevated FSH and LH, average intelligence, and normal androgenization, but his stature was not increased, compared with his father's and brothers'. The i(Xq), found in all cells analyzed, was late-replicating, monocentric, and also asymmetric for the RBG-banding of the two arms, indicating a different chronology of DNA synthesis in each arm. When indicated, in the seven previously reported cases, the level of plasma testosterone was always subnormal; it was normal (650 ng/100 ml) in our patient, who had normal masculinization. Thus the level of testosterone among patients with KS is not necessarily lower with an extra Xq. Furthermore, the sharp contrast in the height of KS patients with or without an i(Xq) is striking. It appears definitely possible to associate the isochromosome Xq Klinefelter syndrome with a lack of height increase.

Adult↗
PubMed1989

Synchronization of amniotic fluid cells for high resolution cytogenetics.

A high resolution technique was applied to amniotic fluid cells by synchronization. After inoculation, the cells were incubated for 30 h in the presence of either thymidine or 5-bromodeoxyuridine (BrdU). After removal of the blocking agent and addition of a low concentration of thymidine, the cells were incubated for another 6 1/2 7 h, then harvested in prometaphase without colcemid. This technique gives a mitotic index of 3.7 per cent after thymidine synchronization and of 3.2 per cent after BrdU synchronization, and more than half of the mitoses were in the earlier phases with the chromosomes showing more than 550 bands per haploid set. GBG, GTG, and RHG prometaphases are presented. Precise high-resolution banding of chromosomes of amniotic fluid cells can increase diagnostic accuracy.

Amniotic Fluid↗
PubMed1989

Dynamic G- and R-banding of human chromosomes for electron microscopy.

Synchronized human lymphocytes were exposed to 5-bromo-2'-deoxyuridine (BrdUrd) for incorporation in either G- or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G- or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R-band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.

Antibodies, Monoclonal↗
PubMed1989

DNA denaturation for ultrastructural banding and the mechanism underlying the fluorochrome-photolysis-Giemsa technique studied with anti-5-bromodeoxyuridine antibodies.

G- and R-bands produced by an immunochemical approach were studied by electron microscopy (EM) to evaluate the role of DNA denaturation on banding quality. Excellent banding was observed only after adequate denaturation by HCl, NaOH and formamide, used in appropriate concentrations to provide uniform 5-bromodeoxyuridine (BrdUrd) exposure by generating single-stranded DNA. Formamide treatment resulted in less intercellular variability. High temperature and high concentrations of NaOH and HCl altered chromosomal morphology. Besides formamide, Hoechst 33258 prestaining which does not interfere with the binding of the anti-BrdUrd antibody and UV irradiation associated with formamide also produced high quality banding. On the other hand, consecutive Hoechst and UV treatment completely inhibited the immunochemical banding. The data indicate that Hoechst and UV act synergistically to disintegrate BrdUrd-substituted chromatin from which DNA is then extracted, leaving only the unsubstituted DNA stainable with Giemsa.

Animals↗
PubMed1989

First cytogenetic evidence of homozygosity for the retinoblastoma deletion in chromosome 13.

Retinoblastoma (Rb) provided the first model in which both normal alleles of a recessive gene had to be lost as a prerequisite for tumorigenicity. However, region q14 of chromosome 13 appears cytogenetically normal in the majority of Rb tumors, and no homozygotic deletion had previously been observed. High-resolution cytogenetics of an Rb tumor revealed a homozygotic deletion in bands q13.3-14.2 of chromosomes 13 in 35% of the cells and a heterozygotic deletion, involving the same region, in 17%. In one third of the cells, although random chromosome loss occasionally occurred, no specific anomaly was detected. The remaining cells showed either monosomy 13, tetraploidy, or an i(1p). Revealing a homozygotic deletion in subbands 14.1 and 14.2 of chromosomes 13 provides the first cytogenetic evidence of the two somatic mutations considered essential to inactivate the Rb gene. These results allow insight into the succession of events necessary for tumor development.

Chromosome Banding↗
PubMed1989

High-resolution R-banding at the 1250-band level. II. Schematic representation and nomenclature of human RBG-banded chromosomes.

Detailed characterization of the RBG-banding pattern at the 1250-band level has been done after thymidine synchronization and block release with 5-bromo-2'-deoxyuridine (BrdU), which induces chromosome elongation and improves definition of chromosomal bands. Optimal conditions for the incorporation of BrdU and the use of the FPG (fluorochrome-photolysis-Giemsa) technique produced excellent band separation and band contrast even in highly elongated prophase chromosomes. Moreover, we did not observe lateral asymmetry in C-banded regions. The schematic representation of these elongated chromosomes in the 1250-band range per haploid set was prepared showing the relative position, the specific size, and the characteristic staining intensity for each band. To this idiogram was extended the International Standard Cytogenetic Nomenclature. This realistic idiogram should help in the preparation of R-banded prophase karyotypes and in the identification and localization of chromosomal rearrangements. Because differences exist between RBG and RHG bands, a brief comparative description of each RBG-banded chromosome is included. Moreover, a minute analysis of the banding pattern revealed that various parts of chromosomes contract differently. We also observed the presence of R-positive bands in heterochromatic regions of the short arms of the acrocentrics, and of chromosomes 1, 9, 16, and Y.

Adult↗
PubMed1989

Physical mapping of probes proximal to the fragile X locus (FRAX) confirms the order F9-DXS105 (cX55.7)-DXS98 (4D8)-FRAXA.

A woman with an abnormal karyotype, (46,X,der(X) (pter----q27::q27----q21), was analyzed using DNA probes in the region Xq27----qter. The results indicate that she is trisomic for the Factor IX locus, disomic for the locus DXS105 (cX55.7) and monosomic for the loci DXS98 (4D8), DXS52 (St14) and Factor VIII. This confirms the absence of the region Xq28 in the abnormal chromosome. Furthermore, the presence of only one copy of 4D8 and two copies of cX55.7 places the DXS98 locus distal to Factor IX and closer to the fragile X locus than DXS105.

Chromosome Deletion↗
PubMed1989

Electron microscopy of gold-labeled human and equine chromosomes.

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

Animals↗
PubMed1988

Human chromosome banding specific for electron microscopy.

Electron microscopy (EM) provides much higher resolution than that obtained with light microscopy (LM). Until now, however, no chromosome banding procedure specifically adapted for EM was available. To produce an easy and reproducible banding method that would allow accurate chromosome identification, we investigated the applicability of an immunochemical method. BrdU-substituted chromosomal regions can be accurately visualized by applying a monoclonal antibody against BrdU, followed by a gold-tagged secondary antibody. Since BrdU is incorporated during the last part of the S-phase, regions of darkly stained G- and C-bands are substituted. A characteristic C-banding pattern is revealed, and the G-banding obtained is sharp and allows discrimination between subbands. Its similarity with the classical G-banding observed by LM makes it easy to interpret and facilitates karyotyping.

Chromosome Banding↗