Differentiation in vitro of human-mouse teratocarcinoma hybrids.

The mouse embryonal carcinoma (EC) line, PCC4, was used to construct a series of somatic cell hybrids which contain a single or a few human chromosomes. The hybrids all retained the EC phenotype as determined by morphology, expression of SSEA-1, lack of cell surface H-2 antigen and cytokeratin filaments, high alkaline phosphatase levels, the ability to form EC tumors ectopically in nude mice, and the ability to differentiate in response to retinoic acid. Constitutively differentiated cloned lines were derived from retinoic acid-treated hybrid cultures. Several derived lines had a phenotype indistinguishable from that of parietal endoderm cells, which includes synthesis of large amounts of laminin, type IV procollagen, and plasminogen activator. One differentiated line showed a fibroblast-like morphology. The differentiated lines derived from two of the hybrids, MCP6 and GEOC4, stably maintained the sole human chromosomal component present in the EC progenitors. These EC hybrids therefore provide a system to study developmental regulation of the introduced and stably maintained human genetic material derived from a variety of cell types.

Human glyceraldehyde-3-phosphate dehydrogenase: mRNA levels and enzyme activity in developing muscle.

Analysis of human glyceraldehyde-3-phosphate dehydrogenase mRNA revealed that levels in adult skeletal muscle are 12-fold greater per microgram of polyadenylated RNA than in fetal skeletal muscle, whereas in cardiac muscle RNA levels were about equal in fetal and adult tissue. The mRNA levels correlate well with glyceraldehyde 3-phosphate dehydrogenase enzyme activities. There was no evidence for fetus- or tissue-specific forms.

Alkaline phosphatase activity in human bladder tumor cell lines.

The cellular localization and isoenzyme pattern of alkaline phosphatase in five cell lines derived from human bladder carcinomas (T24, RT4, RT112, J82, EJ) shown not to be HeLa cells has been established. RT112 cells had a high level of alkaline phosphatase. RT4 had a moderate amount of alkaline phosphatase but in the other three lines, levels were extremely low. Prednisolone caused a small (2 to 3-fold) increase in total alkaline phosphatase in T24 and RT112 lines only. Electrophoretic separation of isoenzymes showed that RT112 and RT4 cells (derived from more highly differentiated tumor types) had three heat stable bands equivalent to placental alkaline phosphatase and three slower bands of a modified placental type. Prednisolone increased only the former. In T24 cells the enzyme resembled the liver-type alkaline phosphatase in electrophoretic mobility and sensitivity to heat denaturation. Cytochemical studies confirmed the presence of cell surface-associated extramembraneous placental type enzyme in RT112 cells. All five cell lines had small deposits of intramembraneous alkaline phosphatase in the plasma membrane and deposits associated tith the mitochondrial membranes and the endoplasmic reticulum that were not completely inhibited by phenylalanine or Levamisole.

Placental-like alkaline phosphatase in malignant and benign ovarian tumors.

Alkaline phosphatase (ALP) components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalanine inhibition and reactivity against antiplacental ALP antiserum. Seven of the eighteen tumors had ALP components which most closely resembled the ALP isoenzyme normally found in placenta and were clearly distinguished from all other tissue ALPs. The proportion of tumors with the placental-like ALP in the malignant group (five out of seven) was significantly greater than the proportion in the benign group (two out of eleven). The fraction (78%) of the malignant tumors with the isozyme represents a larger percentage than has previously been found by examination of cancer patients' sera. The electrophoretic mobilities of the placental-like ALPs in the tumors were in no case identical to the mobilities of any of the six common placental ALP phenotypes. The tumor ALPs may thus be determined by rare variant alleles at the ALP locus, or alternatively, the enzyme molecules may have been subject to structural modification. At least two of these tumors contained an electrophoretically slow. heat-stable, leucine-sensitive ALP, which may correspond to what has been termed the D-variant of placental ALP found in some other tumors.

Use of Alu-PCR to characterize hybrids containing multiple fragments and to generate new Xp21.3-p22.2 markers.

Irradiation fragment hybrids potentially provide highly enriched sources of region-specific human DNA. However, such hybrids often contain multiple human pieces, not all of which can be easily detected. To develop specific resources for rapidly generating markers from Xp21.3-p22.2, we have single cell cloned two previously constructed irradiation hybrids that contain markers in this region and have achieved segregation of the different known fragments originally retained. Alu-PCR products were generated from subclones positive or negative for Xp21.3-p22.2 markers, and comparison of the ethidium bromide patterns between sister subclones facilitated identification of bands likely to map to particular regions; in contrast, subclones that shared markers but were derived from independent lines showed no overlap in ethidium bromide pattern. All Alu-PCR products from one subclone, 50K-19E, in which only three closely linked markers were detected (DXS41, DXS208, DXS274) were mapped back to their region of origin. Of 28 products, 15 mapped to Xp21.2-p22.2, and these make up a new set of regionally assigned markers. However, the mapping data identified four separate Xp fragments in 50K-19E, only one of which had been picked up by marker analysis. Mapping back gel-isolated Alu-PCR products from an irradiation hybrid prior to any cloning or screening generates a comprehensive profile of the human DNA retained and permits rapid selection of sequences derived only from the region of interest.

Human cell lines expressing intestinal alkaline phosphatase.

At least three loci determine human alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]: one coding for the placental form of the enzyme, at least one coding for the intestinal forms, and at least one for the liver, bone, and kidney forms. The alkaline phosphatase in cell line D98/AH-2 has been characterized by inhibition, thermostability, and electrophoretic studies. It is intestinal in type and resembles the fetal intestinal form somewhat more closely than the adult intestinal form. Intestinal alkaline phosphatase was found in the related cell lines Detroit 98, D98/S, and D98/AH-R. No placental alkaline phosphatase could be detected in any of these cell lines. This series of cell lines are believed, on the basis of earlier investigations, to be HeLa in origin but other HeLa cell lines show placental alkaline phosphatase. Loss of expression of the placental alkaline phosphatase locus probably occurred prior to the separation of Detroit-98 from the lineage leading to other HeLa cell lines and this has persisted in the Detroit-98 derivatives D98/AH-2, D98/S, and D98/AH-R. Another possibility is that placental alkaline phosphatase expression only appeared in the HeLa lineage subsequent to the separation of Detroit-98.

Cytogenetic and molecular analysis of sex-chromosome monosomy.

X chromosome- and Y chromosome-specific DNA probes were used to study different aspects of the genesis of sex-chromosome monosomy. Using X-linked RFLPs, we studied the parental origin of the single X chromosome in 35 spontaneously aborted and five live-born 45,X conceptions. We determined the origin in 35 cases; 28 had a maternal X (Xm) and seven had a paternal X (Xp). There was a correlation between parental origin and parental age, with the Xp category having a significantly reduced mean maternal age by comparison with the Xm group. Studies aimed at detecting mosaicism demonstrated the presence of a Y chromosome or a second X chromosome in three of 33 spontaneous abortions, a level of mosaicism much lower than that reported for live-born Turner syndrome individuals.

Members of the human glyceraldehyde-3-phosphate dehydrogenase-related gene family map to dispersed chromosomal locations.

Several highly homologous glyceraldehyde-3-phosphate dehydrogenase (GAPD)-related sequences have been identified previously in human DNA by Southern blot analysis. Protein studies have identified only a single expressed locus for this major glycolytic enzyme, and this maps to chromosome 12p13. Sequence analysis of a GAPD muscle cDNA clone and a GAPD-related clone retrieved from an X-chromosome recombinant library showed that the latter was a processed pseudogene that maps to Xp11-p21. In this study, we have determined the chromosomal locations of several of the additional GAPD-related human sequences using a short 3' end sequence from the cDNA to probe DNA from a series of human-rodent somatic cell hybrids on Southern blots. Eight HindIII GAPD-related sequences detected at high stringency have been mapped to 6 different chromosomes. Several of the additional sequences detected at more moderate stringency have been localized to a further 10 chromosomal sites. Together, these sites constitute the known expressed locus, the known X-linked pseudogene, and 15 GAPD-like loci.

Modulation of the expression of alkaline phosphatase genes in a human malignant cell line passaged through nude mice.

Hep 2/5 cells, a clone of HeLa which expresses both placental-type alkaline phosphatase and fetal intestinal alkaline phosphatase (ALP), were grown as solid tumors in nude mice. Analysis of ALP isozymes present in tumor specimens excised at intervals over a 52-day period showed that levels of placental-type ALP had dropped markedly, while levels of the intestinal ALP remained fairly constant. Cells re-cultured from two separate tumors regained most of the original placental ALP levels. However, levels of the fetal intestinal ALP in cells returned to culture rapidly increased to more than six times the levels in the original cultured cells, and these increased levels persisted. Passage through nude mice thus induced at least semi-permanent changes in the level of expression of fetal intestinal ALP in Hep 2/5 cells, and the nature of these changes may relate to the role of the isozyme locus in malignant cells.

Alkaline phosphatase expression in human cell lines derived from various malignancies.

A search for expression of heat-stable placental-type alkaline phosphatase (ALP) has been carried out in 19 unselected human tumor cell lines, known not to be HeLa. All cell lines showed measureable ALP activity and in 15 of the lines at least low levels of a heart-stable, presumptively placental-type ALP were detected. In five of these lines where the level of this heat-stable activity was sufficient, further, investigation, (immunologic, inhibition and electrophoretic studies) demonstrated that this ALP was placental-type in its characteristics and clearly different from liver/bone/kidney or intestinal ALPs. In 10 lines the heat-stable activity was too low to allow further characterization, In four lines no heat-stable activity in these various lines was liver-bone-kidney in type. This study suggests that the placental ALP locus may be expressed in at least at low levels in a much higher proportion of tumors and tumor cell lines than previously reported. The findings taken together with recent reports that low levels of placental-type ALP are present in some normal adult tissues (cervix, Goldstein et al., 1980; testis, Chang et al., 1980), indicate that so-called "ectopic" synthesis of placental ALP in tumor cells may not necessarily be due to derepression of a structural locus which is completely unexpressed in normal adult tissues. It may represent an enhancement of expression in malignancy or there may be clonal expansion of a particular cell type which normally expresses the alkaline phosphatase at a high level.

Heterogeneity of alkaline phosphatases in different HeLa lines.

Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALP structural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the ""derepressed'' placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.

Alkaline phosphatase phenotypes in tumour and non-tumour cell lines: not an invariable marker for neoplastic transformation.

The cytochemical localisation and presumed isoenzyme type (based on selective inhibition experiments) of alkaline phosphatase in 5 cell lines derived frrom normal human, rat, mouse and hamster tissues, 6 human lymphoblastoid lines and 6 human and mouse tumour-derived cell lines are described. Enzyme activity varied between the cell lines. An isoenzyme inhibited by L-phenylalanine was present in 3 normal lines, 3 lymphoblastoid lines and 2 tumour lines. The presence of this isoenzyme cannot be used as a marker of neoplastic transformation.

Suppression and reexpression of human intestinal-like alkaline phosphatase in intraspecific hybrids.

Expression of the gene locus which codes for a form of human intestinal alkaline phosphatase (ALP) has been analyzed in intraspecific somatic cell hybrids. Hybrids were constructed between D98/AH-2, a line of HeLa which ectopically synthesizes high levels of this ALP isozyme, and three different nonintestinal ALP-producing diploid lines. In chromosomally complete hybrids, expression of the ALP isozyme was initially suppressed, but on extended culture, reexpression occurred, as did limited chromosome loss. Results from extensive subcloning experiments showed that events leading to reexpression occurred at high frequency, and this ALP reexpression appeared to confer some selective advantage, direct or indirect, on the cells. In the fibroblast hybrids, reexpression of the intestinal-like ALP was always accompanied by new, high-level expression of liver/bone/kidney ALP, the product of a separate ALP gene locus. Thus expression of the one ALP locus is not excluded and, in fact, appears to be promoted by expression of the other in these cells.

A glyceraldehyde-3-phosphate dehydrogenase pseudogene on the short arm of the human X chromosomes defines a multigene family.

A human X chromosome-derived gene sequence which recognises an abundant, 1.2-kb mRNA in several cell types was previously isolated during a study to identify expressed sequences from an X chromosome recombinant library. Further characterisation of this clone, acronym OA1, has shown that it maps to the short arm of the X, at Xp21 to Xp22. A 777-bp fragment of the clone which hybridizes to the 1.2-kb mRNA has been sequenced, and the inferred amino acid sequence shows 80% homology with the published protein sequence for human muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fragment shows even higher homology (87%) with pig muscle GAPDH. The OA1 clone selects an mRNA which translates in vitro into a polypeptide of 36 K, the subunit size of GAPDH. However, the X-sequence is most probably a pseudogene whose structure is consistent with it having arisen by reverse transcription of a GAPDH or GAPDH-related mRNA followed by insertion into the X chromosome. The GAPDH-related portion of OA1 hybridizes to several DNA fragments in human and mouse DNA, and six fibroblast cDNA clones which cross-hybridize to OA1 identify the same genomic fragments as OA1. This series of clones identifies a new, conserved GAPDH-related multigene family.

Human-mouse hybrids with an embryonal carcinoma phenotype continue to transcribe HLA-A,B,C.

We previously constructed a hybrid cell line, MCP6, which contains an X/6 translocation chromosome as its sole human genetic component in a mouse embryonal carcinoma (EC) cell background. This chromosome, which carries the major histocompatibility complex (MHC) originated from a human B cell which expresses class I and class II MHC antigens. EC cells do not express class I or class II antigens on their cell surface. Northern blot analysis has now shown that in the MCP6 hybrid, human class I genes, i.e., HLA-A,B,C, continued to be transcribed, and cellular levels of the transcripts were similar to, or only slightly lower than, levels in hybrids with a non-EC phenotype. However, very low levels of mRNA species recognised by a mouse class I gene (H-2) probe were also detected in EC cells and EC hybrids. Comparison of the relative levels of H-2 and HLA class I gene transcripts in the EC hybrids and non-EC hybrids indicated that the introduced HLA-A,B,C genes were not appropriately regulated in the EC cell but were subject at least in part to cis control. In contrast to the class I genes, no class II gene (i.e. HLA-DR alpha) transcripts were detected in MCP6. Hybrid EC lines thus provide a system to investigate the different levels of control of MHC gene expression during development and may help to elucidate mechanisms whereby the embryonic genome programs expression of differentiated cell functions.

Identification and regional localization of a highly polymorphic dinucleotide repeat D11S614 to the interval in 11q23.3 flanked by recurrent translocation breakpoints.

A highly informative dinucleotide repeat polymorphism has been identified at the D11S614 locus on chromosome 11q23. Ten different alleles have been observed at this locus, and the heterozygosity frequency is approximately 85%. Physical localization of this marker in a panel of somatic cell hybrids containing chromosome 11 translocations showed that it maps to 11q23.3, within the interval between the recurrent t(4;11) leukaemia breakpoint and the t(11;22) Ewing's sarcoma breakpoint. This physical mapping data is consistent with the genetic mapping which indicates tight linkage to other markers in the q23.3 region including PBGD, CD3D and D11S29. Regional localization of highly informative markers such as D11S614 will facilitate integration of the genetic and physical maps.

Physical mapping of 38 highly informative genetic markers to 10 intervals of chromosome 11q: integration of the physical and genetic maps.

A large number of highly polymorphic microsatellite markers particularly suitable for genetic linkage analysis have recently been developed. In order to facilitate integration of the genetic maps of chromosome 11q generated using these types of markers with the physical maps of 11q currently being assembled, we have regionally assigned the Genethon markers and the 11q designated index plus other commonly used polymorphic markers to ten physical intervals of 11q. These intervals are defined by translocation breakpoints immortalized in somatic cell hybrid lines and can therefore serve as readily accessible and stable landmarks for detailed map integration and facilitate the derivation and placement of new markers and cloned contigs.

A comparative study of eight cell lines derived from human testicular teratocarcinoma.

Eight independent cell lines, derived from human testicular germ-cell tumors, were examined for the expression of various markers. These included major histocompatibility and embryonic antigens, chorionic gonadotropin, alpha fetoprotein, alkaline phosphatase, plasminogen activator, and infectivity by SV40. No line consisted primarily of choriocarcinoma or yolk sac cells, but several contained cells resembling murine embryonal carcinoma; some of these lines formed tumors with the distinctive features of embryonal carcinoma when injected into immunosuppressed animals. It is proposed that human embryonal carcinoma cells, unlike those of the mouse, correspond to a preblastocyst stage of development.

Alkaline phosphatase in mitochondria.

In electron microscope cytochemical studies alkaline phosphatase activity was present in the mitochondria of all liver cells and associated with the plasma membrane of the cells of bile canaliculi. The mitochondrial activity was partially inhibited by L-phenylalanine and Levamisole but the plasma membrane associated activity was completely inhibited by Levamisole. Biochemical assays have shown that a significant amount of the total mouse liver alkaline phosphatase activity was present in the mitochondria fraction. Starch gel electrophoresis showed that this mitochondrial alkaline phosphatase had a characteristic isoenzyme pattern, consisting of 3 distinct bands which were not retarded by neuraminidase treatment. The enzyme in the mitochondria-free supernatant showed one wide band which was retarded by neuraminidase.

A method for generating hybrids containing nonselected fragments of human chromosomes.

We have used an irradiation and fusion technique to generate somatic cell hybrids that contain human chromosomal fragments. As a model system, a human-hamster hybrid containing a single human X chromosome was gamma-irradiated and fused with a rodent line. Hybrids were obtained without imposing direct selection for human material. Analysis of 29 clones by in situ hybridization and Southern blotting revealed that human fragments were incorporated into the hybrid cell genomes in most lines. Like chromosome-mediated gene transfer (CMGT)-generated hybrids, these hybrids contained multiple human fragments and retained alphoid centromeric sequences with a high frequency. However, unlike the CMGT, human fragments (apart from alphoid sequences) of less than 10(7) bp showed no evidence for rearrangements. This technique provides a method for constructing hybrids that contain a limited number of small human fragments derived exclusively from any chromosome of choice without the need to impose selection. Such hybrids provide a valuable resource for high-resolution mapping over short distances and for the isolation of disease and other loci mapped genetically.