Isozyme pattern in serially xenotransplanted childhood tumors.

Growth rate, histological course, and polymorphic enzyme pattern (glucose 6-phosphate dehydrogenase, glucose phosphate isomerase, and phosphofructokinase) were studied in eight childhood tumors xenotransplanted serially to nude mice. The growth rate of these tumors (three nephroblastomas, one hypercalcemic renal tumor, three rhabdomyosarcomas, and one malignant histiocytosis) appeared stable for any one particular tumor line. The time interval between two grafts varied from 1 to 3 weeks to 1 to 2 months in correlation with the clinical course of each malignant process. Histological changes were mostly in relation with a progressive dedifferentiation of the grafts. Immunoneutralization of glucose-6-phosphate dehydrogenase and glucose phosphate isomerase made possible the quantification of the stroma reaction in the grafts. A series of ten passages showed the amount of stroma to be constant for a given tumor type but variable from one tumor type to another, except for the malignant histiocytosis which showed an increase in stroma constituent after the sixth passage. One nephroblastoma tumor line showed, during the third passage, a sudden acceleration in the growth rate and complete transformation of the histological and isozymic patterns, which were interpreted as being the result of a murine lymphoma. The fibroblastic form of phosphofructokinase increased in every tumor line, whatever the tumor type. This change may be linked to a progressive dedifferentiation during the passage.

hSNF5/INI1 inactivation is mainly associated with homozygous deletions and mitotic recombinations in rhabdoid tumors.

The chromatin-remodeling hSNF5/INI1 gene has recently been shown to act as a tumor suppressor gene in rhabdoid tumors (RTs). In an attempt to further characterize the main chromosomal mechanisms involved in hSNF5/INI1 inactivation in RTs, we report here the molecular cytogenetic data obtained in 12 cell lines harboring hSNF5/INI1 mutations and/or deletions in relation to the molecular genetic analysis using polymorphic markers extended to both extremities of chromosome 22q. On the whole, mitotic recombination occurring in the proximal part of chromosome 22q, as demonstrated in five cases, and nondisjunction/duplication, highly suspected in two cases (processes leading respectively to partial or complete isodisomy), appear to be major mechanisms associated with hSNF5/INI1 inactivation. Such isodisomy accompanies each of the RTs exhibiting two cytogenetically normal chromosomes 22. This results in homozygosity for the mutation at the hSNF5/INI1 locus. An alternate mechanism accounting for hSNF5/INI1 inactivation observed in these tumors is homozygous deletion in the rhabdoid consensus region. This was observed in each of the four tumors carrying a chromosome 22q abnormality and, in particular, in the three tumors with chromosomal translocations. Only one case of our series illustrates the mutation/deletion classical model proposed for the double-hit inactivation of a tumor suppressor gene.

Pro-opiomelanocortin gene expression in human phaeochromocytomas.

Phaeochromocytoma is an occasional cause of the ectopic ACTH syndrome. The mechanisms of proopiomelanocortin (POMC) gene expression were analysed in 11 human tumours not associated with Cushing's syndrome, by detecting and characterizing the POMC mRNA. A DNA probe corresponding to most of the protein-coding region of the third exon was used in Northern blot studies of total and poly(A)+ RNA. All tumours contained a short (800 bases) mRNA species different from the 1200 base mRNA species of the human pituitary. This short mRNA was also present in the normal adrenal, where S1 mapping showed that it resulted from transcription initiation within the third exon. However, in two tumours, equivalent amounts of the 1200 base mRNA were also present, and in one of them a third POMC mRNA of approximately 1450 bases was detected. These data show that POMC gene expression occurs in all phaeochromocytomas. It is suggested that excess production of the 1200 bases (or the larger, 1450 base) mRNA in some tumours may be responsible for the rare occurrence of the ectopic ACTH syndrome.

Cell differentiation in Wilms' tumor (nephroblastoma): an immunohistochemical study.

Using an indirect immunoperoxidase technique, we tested frozen specimens from 12 Wilms' tumors with monoclonal antibodies (MoAbs) reacting against a large panel of molecules including laminin, fibronectin, cytokeratin, vimentin, villin, CD24, CALLA/CD10, CR1, CD26, class I and class II major histocompatibility complex (MHC) molecules, and endothelium factor VIII. These molecules were chosen because they are markers of specific segments of the mature kidney and because their loss or acquisition is indicative of different steps of human nephrogenesis. KI67 MoAb was used to evaluate the proliferating activity of the cells. The blastemal component (cell compact areas) of Wilms' tumors consisted of vimentin-positive cells with a fibronectin network. However, signs of epithelial maturation were present in compact areas where cytokeratin-positive cells producing laminin were observed. The cells exhibited a high degree of proliferating activity. The tubule formations consisted of cytokeratin-positive cells and had a defined laminin border. All the cells, whether in compact areas or in tubules, were strongly CD24-positive. Some tubular formations showed signs of proximal maturation with the presence of CALLA, CD26, and even villin. In four cases class I-MHC molecules were expressed by some tubular cells. Large cystic cavities present in five cases were edged by cytokeratin, CD24-positive cells, or by vimentin, CALLA, CR1-positive cells. Some glomeruloid bodies, present in two cases, were also composed of vimentin, CALLA, and CR1-positive cells which correspond to the mature podocyte phenotype. The interstitial tissue contained mainly laminin and fibronectin network with macrophages and few CD3 lymphocytes. The presence of large cells with muscular differentiation was noted; round vimentin and CD26-positive cells were also seen. The endothelial cells of the vessels exhibited vimentin, factor VIII, and class I and class II MHC molecules as do mature cells, but in some cases the endothelial cells lacked class II molecule expression and were CALLA-positive. These results which confirmed and extended those previously described show that cell differentiation in Wilms' tumor mimics that observed during metanephros development. Moreover, this study shows that tumoral cells in nephroblastoma share several antigens with cells from lymphoid lineage (CD24, CALLA, and CD26) as do developing and mature kidney cells. Such cell phenotype dissection provides a useful and reliable tool for testing the influence of various factors on the development of hetero-transplanted or cultured Wilms' tumors.

Ectopic G-29 and G-37 glucagon secretion by hypercalcemic infantile renal tumors.

Four hypercalcemic infantile renal tumors were shown to secrete glucagon-like peptides. These unusual tumors were histologically classified as rhabdoid tumors of the kidney (3 cases) and a cellular mesoblastic nephroma (1 case). Elevated G-29 and G-37 glucagon levels were detected in the plasma and tumor extracts as well as in the supernatants of cultured tumor explants. Three of these tumors were heterotransplanted into the nude mice and serially passaged from a mouse to another. The glucagon level decreased in the transplanted tumor extracts with the number of passage.

Cytogenetic study of cell lines from an infantile hypercalcemic renal tumor.

Four cell lines were obtained in vivo and in vitro from an infantile hypercalcemic renal tumor, which is considered to be a new tumor entity. Biochemical characteristics of the cells, studied after heterotransplantation into nude mice, were similar to those observed in the original tumor. One of the two in vitro cell lines originated from the initial tumor, the other from transplanted tumors in nude mice. One of the two in vivo cell lines originated in nude mice from serial grafts of the initial tumor, the other from grafts of the first in vitro cell line. All cell lines showed a human diploid karyotype, except for the cell line obtained directly from the tumor. In the latter, the karyotypes showed either a regional duplication of the long arm of chromosome #5 or a duplication of the long arm of chromosome #21. These two rearrangements did not appear simultaneously in the same cells, and their frequencies changed at each passage. The study of these different cell lines showed a remarkable karyotype stability, which did not prevent successful grafting into nude mice.

Reactivity of histiocytosis X cells with monoclonal antibodies.

Histiocytosis X cells were demonstrated to react with T6 antigen as well as with the M1 and I1 markers of monocytes using immuno-electron microscopy and double labeling immunofluorescence technique. The data confirm the close relationship existing between histiocytic X cells, Langerhans cells and dendritic cells, and suggest to consider the T6 antigen either as an early differentiation marker of thymocytes or as a functional marker of Mononuclear Phagocyte System subpopulations.

Infantile renal tumors associated with hypercalcemia. Characterization of intermediate-filament clusters.

Three cases of hypercalcemic infantile renal tumors were studied to identify the proteins related to their cytoplasmic 8- to 10-nm filament clusters. Immunofluorescence microscopy, using antibodies against vimentin, cytokeratin, and actin, led to the characterization of vimentin-positive filament clusters. We discuss the possibility of a mesenchymal origin for these tumors.

[Dynamic studies of Langerhans cell histiocytosis cells. Their contribution to the current concept of the disease. Report of a series of 38 cases]. / Etudes dynamiques des cellules de l'histiocytose Langerhansienne. Leur apport dans le concept actuel de la maladie. A propos d'une série de 38 observations.

In vitro explantation of 38 fragments of eosinophilic granuloma of bones was attempted. A satisfactory growth was obtained in nearly 90% of cases. This short-term culture maintained the ultrastructural characteristics and, to a lesser extent, the cytochemical features of the Langerhans cells, confirming the Langerhans cell origin of this cell proliferation. In addition, this procedure was able to demonstrate an immunodependent erythrophagocytosis (3/3) and a preferential fixation of labelled precursors (Glycerol, choline of lipid metabolism as well as labelled dopamine (2/2). All attempts to obtain a permanent cell line and graft to nude mice (even irradiated) failed. Under the in vitro conditions, the Langerhans cells do not divide up but can be readily identified up to 2 or 3 weeks. The contrast between the evident in vivo proliferation and the in vitro quiescent state suggests that some undetermined growth-factors targeted to the Langerhans cell system are missing in our commonly used culture media. The in vitro culture procedure could be of some help to their identification.

Ig surface receptors and erythrophagocytic activity of histiocytosis X cells in vitro.

Membrane receptors for Ig and IgG and receptors for immune complexes (C3) have been identified on the surface of histiocytosis X cells by direct immunofluorescence and by rosette formation. Histiocytosis X cells also avidly phagocytose human RBC and latex particles. The cells studied originated from three eosinophilic granulomas of bone and were maintained in culture for 12 days. These immunological characteristics suggest that histiocytosis X cells represent a neoplastic variant of a cellular subpopulation (Langerhans lineage) which probably belongs to the mononuclear phagocyte system.

The ras-related ral gene maps to chromosome 7p15-22.

Human cDNAs coding for the new protein ral that shares 50% homology with the ras proteins have been recently isolated. A 600-bp fragment carrying mainly the coding region was used to localize the ral gene by hybridization with sorted chromosomes and in situ hybridization. Direct molecular hybridization on sorted chromosomes using a single laser illumination allowed the assignment of the ral gene to a region of the flow karyotype containing chromosomes 7, 8 and X. With dual laser analysis ral was assigned to the fraction containing chromosome 7. In the 331 human metaphases hybridized with the 3H-labelled insert, the silver grain distribution showed a unique major signal on chromosome 7p15-22.

In vitro behavior of human fetal lung maintained in organ culture. Light and electron microscopic studies.

Human fetal lung obtained from 9-26 = weeks = old embryos were maintained in organ culture for 2-5 weeks. The in vitro survival and changes are clearly age dependent. The best survival was obtained with lung tissue from the early glandular period. With these young embryos tubular dilation was frequent during the 1st week. The relatively short duration of culture permitted only a fragmentary study of differentiation of the human lung in vitro but, with the exception of tubular dilations, most of the in vitro changes were also found during lung differentiation in vivo = monostratification of epithelium, bronchiolar development, decrease of glycogen, appearance of myelinlike figures, fibroblastic and myoblastic transformation of mesenchymal cells.

The chromosomal localization of the human follicle-stimulating hormone receptor gene (FSHR) on 2p21-p16 is similar to that of the luteinizing hormone receptor gene.

Two cDNA probes (5' and 3' region) corresponding to the human follicle-stimulating hormone receptor gene (FSHR) were used for chromosomal localization by in situ hybridization. The localization obtained on chromosome 2p21-p16 is similar to that of the luteinizing hormone/choriogonadotropin (LH/CG) receptor gene.

Chromosomal localization of the type-I 15-PGDH gene to 4q34-q35.

The gene encoding the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase, designated type-I 15-PGDH, was mapped to chromosome 4 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This gene was further localized to bands 4q34-q35 by in situ hybridization on human chromosomes.

Chromosomal localization of two human zinc finger protein genes, ZNF24 (KOX17) and ZNF29 (KOX26), to 18q12 and 17p13-p12, respectively.

Two members of the zinc finger Krüppel family, ZNF24 (KOX17) and ZNF29 (KOX26), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization to human chromosomes 18q12 and 17p13-p12, respectively. The mapping of ZNF29 together with the previously reported localization of ZFP3 suggests that a zinc finger gene complex is located on human chromosome 17p. ZNF29 maps centromeric to the human p53 tumor antigen gene (TP53). In the analogous murine position, the two mouse zinc finger genes Zfp2 and Zfp3 have recently been assigned to the distal region of mouse chromosome 11, the murine homolog of human chromosome 17. Both human zinc finger genes ZNF24 and ZNF29 are in chromosomal regions that have been noted to be deleted in neoplasms of the lung and of the central nervous system at chromosome 17p and in colorectal neoplasia at chromosomes 17p and 18q.

Chromosome assignment of four RAS-related RAB genes.

The human RAB genes share structural and biochemical properties with the RAS gene superfamily. The encoded RAB proteins show 38 to 75% amino acid identity with the yeast YPT1 and SEC4 gene products. We used four human RAB-cDNAs, RAB3B, RAB4, RAB5 and RAB6, to map the corresponding genes on human chromosomes. These genes were assigned to 1p32-p31, 1q42-q43, 3p24-p22 and 2q14-q21, respectively, by in situ hybridization.

Two human genes encoding zinc finger proteins, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), map to chromosome 7p22-p21 and 12q24.33, respectively.

Two members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.

Chromosomal localization of two KOX zinc finger genes on chromosome bands 7q21-q22.

Human cDNAs encoding Krüppel-type zinc finger domains, designated KOX 1-32, have been cloned from human T lymphocyte cell line libraries. We report here the regional localizations by in situ hybridization of KOX 18 and KOX 25 on chromosome bands 7q21q22. Pulse-field gel electrophoresis (PFGE) analysis showed that these genes are physically located within a DNA fragment of 250 kb. The genes KOX 4 and KOX 9, mapped on chromosome 8q24, were found to be located within a DNA fragment of 450 kb. From the present and previous data, eighteen different KOX genes have been located at least two by two within nine DNA fragments of 200 to 580 kb.

Bilateral nephroblastoma associated with a 3;17 translocation.

Cultured cells from the tumor of a child with bilateral nephroblastoma were studied cytogenetically. All mitoses observed showed the same male karyotype, 46,XY,t(3;17). This translocation constitutes a newly discovered rearrangement that has not been reported previously either in nephroblastoma or in other neoplastic processes.