Six loci mapped on to human chromosome 2p are assigned to sheep chromosome 3p.

Six loci, apoliproprotein B (including Ag(x) antigen), immunoglobulin kappa constant region (IGKC), luteinizing hormone/choriogonadotrophin receptor, avian myelocytomatosis viral related oncogene, neuroblastoma derived, ornithine decarboxylase, and proopiomelanocortin (adrenocorticotropin/beta-lipotropin) (POMC), were newly assigned to sheep chromosome 3p using a chromosomally characterized minipanel of sheep-hamster cell hybrids. Isotopic in situ hybridization of IGKC to sheep chromosome 3p22-p17 is reported, confirming the cell hybrid assignment. As these loci are all known to map to human chromosome 2p, this study demonstrates that this chromosomal segment is extensively conserved in sheep. Only POMC has been previously assigned to cattle chromosome 11, which is the equivalent of sheep chromosome 3p. Therefore, we predict that the other loci assigned in this study to sheep 3p are likely to be located on cattle 11. The provisional assignment of an additional locus, annexin-like to sheep chromosome 3p is also reported.

Malignant melanoma: from subcutaneous nodule to brain metastasis.

A thirteen-year-old patient, diagnosed with melanoma was followed cytogenetically using short-term cultures of specimens from a subcutaneous nodule, lymph node, and brain metastases. Simple hypodiploid karyotypes with loss of heterozygosity for chromosomes 5, 9, 10, 16 and 17 and two structural changes in 3 and between 13 and 21 increased in complexity to near triploid in the lymph node. Two cell types of the lymph node showed a progression of structural rearrangements prior to metastasis to the brain. Translocation of 6p to chromosome 2, deletions of 8, and a translocation (2;19) preceded p and q arm deletions of both chromosomes 9 and 11 in the lymph node. Cells metastasizing to the brain showed the accumulation of all previous aberrations and had acquired a direct duplication of distal 17 long arm. Whether or not elevated levels of protein kinase C, located on chromosome 17q contribute to tumor adhesion and growth on the brain remains to be elucidated. Identification of most chromosomes undergoing rearrangement was carried out using whole chromosome painting probes in in situ hybridizations. Some of these rearrangements would have been impossible to identify by standard karyotyping.

Cytogenetic homogeneity in eight independent sites in a case of malignant melanoma.

Melanoma cell lines initiated from metastases excised at the same time from multiple sites in a patient reflect a single clone origin. The similarities of the karyotypes of these lines depend upon the site of excision, the selective survival in vitro, and the time span in vitro before study. In the present investigation, eight short-term cultures of malignant melanoma from the same patient were analyzed cytogenetically, six after the second passage in vitro, one from the third, and one from the fifth passage. Karyotypes were similar on all eight cultures, each having chromosome abnormalities der(1)t(1q;?), der(3)t(1;3), and t(7;9). Four cell lines had an isochromosome 4q, which was missing in four lines. Two cell lines were missing del(6). In five of eight cultures there was an occasional i(16q) or t(16q;21q). In the final culture, made 6 months after the first seven, three cell types present included a type corresponding to that of seven previous cultures, a similar cell type with an abnormal chromosome 11, and a third cell type with a 3;19 translocation. Comparisons are made with similar studies in the literature and a hypothesis has been formulated to explain melanoma metastases as revealed by the cytogenetics of multiple lesions excised simultaneously.

Mapping the sheep genome: production of characterized sheep x hamster cell hybrids.

Specific chromosomes of sheep have been selectively "captured" in hamster x sheep cell hybrids produced by fusing different Chinese hamster auxotrophs with lymphocytes from sheep carrying normal or Robertsonian translocation chromosomes. A minipanel has been established comprising sheep chromosomes 1; 3; t1 = rob(6;24); t2 = rob(9;10); and X in a predominantly monochromosomal state. Using this targeted cell fusion approach the nutritional markers, uridine monophosphate synthetase (UMPS), mapped onto human chromosome 3 (HSA3), phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (GART) (HSA21), are reported to be located on sheep chromosome 1q; and phosphoribosyl pyrophosphate amidotransferase (PPAT) (HSA4) has been assigned to sheep chromosome 6. By isozyme analysis and Southern hybridization, transferrin (TF) and superoxide dismutase 1 (SOD1), both in the bovine syntenic group U10, were assigned to sheep chromosome 1q; adenylate kinase 1 (AK1), in the bovine syntenic group U16, to sheep chromosome 3p; lactate dehydrogenase B (LDHB) and phenylalanine hydroxylase (PAH) on bovine chromosome 5 (BTA5) to sheep chromosome 3q; phosphoglucomutase 2 (PGM2) (bovine syntenic group 15) to sheep chromosome 6 (t1q); avian myelocytomatosis viral oncogene homolog (MYC) and Moloney murine sarcoma viral oncogene homolog (MOS) (BTA 14) to sheep 9 (t2q); and glutathione reductase (GSR) (bovine syntenic group U14) to sheep 24 (t1p).

Preferential chromosome 11q and/or 17q aberrations in short-term cultures of metastatic melanoma in resections from human brain.

Thirteen specimens of metastatic malignant melanoma resected from eight patients undergoing craniotomy were analyzed cytogenetically from short-term cultures. All patients had chromosome 1 aberrations, as did three of four patients with metastases only to extracranial sites. Both groups had variable involvement of chromosomes 3, 6, 7, and 8. Only those with brain metastases had 11q and/or 17q involvement in six of eight patients. In reported cases of nonbrain metastases, when chromosome 11 was involved, the short arm was usually deleted or replaced through translocation; on the contrary, in reports on patients with brain metastases, the long arm of chromosome 11 was deleted at q23 or was the recipient of a translocation at q23, and/or 17q was present as an isochromosome. These aberrations were similar to those found in the patients with brain metastases in this report. Two patients undergoing brain resections did not show 11q or 17q aberrations, one near diploid with t(10;19) and the other near hexaploid with few structural rearrangements. The neural cell adhesion molecule gene is located near 11q23, and the neural growth factor receptor is located near 17q21-q22. The relevance of these genes to brain metastases in melanoma is under investigation.

Characterization of a continuous human glioma cell line DBTRG-05MG: growth kinetics, karyotype, receptor expression, and tumor suppressor gene analyses.

The establishment of a new glioma cell line, DBTRG-05MG, in a modified RPMI 1640 medium is described. The cells were derived from an adult female with glioblastoma multiforme who had been treated with local brain irradiation and multidrug chemotherapy; the tumor showed substantial change in histologic appearance compared to the original biopsy 13 mo. previously. The line has been successfully cryopreserved and passaged up to 20 times. The karyotype of the cells demonstrated it as a hypotetraploid line; the DNA index of 1.9 confirmed the karyotype analyses. By immunocytochemical analysis, the cell line reacted with polyclonal antibodies to vimentin, S100, and neuron specific enolase, reflecting its primitive neuroectodermal character. Positive immunostaining for epidermal growth factor receptor correlated with the excess of chromosome 7 seen in the karyotype. The cell line reacted negatively to antibodies against platelet-derived growth factor and its receptor, neuronal cell adhesion molecule, and glial fibrillary acidic protein. By flow cytometry, the cells were major histocompatibility class I antigen positive and class I antigen negative. Growth kinetic studies demonstrated an approximate population doubling time of 34 to 41 h and a colony forming efficiency of 71.4%. Western blot analysis showed the presence of low levels of normal-sized retinoblastoma protein. When compared to the patient's lymphocyte DNA, no loss of heterozygosity of the p53 tumor suppressor gene was observed in the DBTRG-05MG cell line DNA.

Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.

The primitive neuroectodermal tumors (PNET) comprise a class of malignant nervous system neoplasms that afflict children. These tumors consist of cells that are morphologically identical to the primitive neuroepithelial cells normally seen in early stages of neural embryogenesis, supporting the notion that PNET result from a disturbance in the process of normal neuronal or glial differentiation. In the central nervous system, PNET occur most commonly in the cerebellum (medulloblastomas), but only occasionally in the cerebral hemispheres. We report here the establishment and characterization of a new human cell line (PFSK) derived from a PNET from the cerebral hemisphere of a child. The growth characteristics of PFSK cells were typical of an immortalized, transformed cell line. Cytogenetic and molecular genetic studies showed that three different sublines were present. In one of these sublines, sequences from chromosome 17 had been lost during establishment in culture. Immunocytochemical studies showed that PFSK cells expressed nestin, an intermediate filament protein normally expressed by neuroepithelial stem cells during neurulation. The PFSK cells did not express antigens typically found in terminally differentiated neurons or glia, indicating that this tumor cell line might represent neuroepithelial stem cells prior to commitment to a neuronal or glial lineage.

Bleeding secondary to the use of thrombolytic agent in a patient with recent renal lithotripsy.

We feel that while our patient succumbed to complications of his extensive anterior myocardial infarction, the devastating renal, subcapsular, and retroperitoneal hemorrhage as well as the cerebral hemorrhage certainly appeared to hasten his demise. From this experience, we now consider recent renal lithotripsy to be a major contraindication to the use of thrombolytic agents. Logically, the time frame for omitting thrombolytic therapy following renal lithotripsy should be similar to that of other major surgical procedures, approximately four to six weeks.

Treatment of t(4;11) acute leukemia with aggressive multiagent chemotherapy. Preliminary results.

Acute leukemia patients with a 4;11 translocation have had a poor prognosis in the past, averaging 9 months survival from diagnosis. Three patients from The Children's Hospital of Denver were reported in 1982--all died. This report describes five additional patients who fared better. Aggressive treatment during induction and maintenance with intensive alternating non-cross-resistant chemotherapeutic agents may have contributed to the improved survival.

Chromosome mapping of biological pathways by fluorescence-activated cell sorting and cell fusion: human interferon gamma receptor as a model system.

Human chromosome 6 encodes both the interferon gamma receptor as well as the class I major histocompatibility complex antigens, HLA-A, -B, and -C. However, the presence of chromosome 6 in somatic cell hybrids is insufficient to confer sensitivity to human interferon gamma (Hu-IFN-gamma) as assayed by class I HLA induction; it is necessary for both human chromosomes 6 and 21 to reside in the hybrid to generate a response to Hu-IFN-gamma. Treatment of such a hamster-human hybrid, Q72-18, with Hu-IFN-gamma induces the class I HLA antigens. Q72-18 cells selected by fluorescence-activated cell sorting for the loss of class I HLA induction also lost human chromosome 21. Fusions of such cells to a hybrid that contains only human chromosome 21 reconstitutes HLA antigen induction by Hu-IFN-gamma. Furthermore, fusions of hybrids containing a translocated human chromosome 6q and the HLA-B7 gene to a line containing only human chromosome 21 or a translocated 21q also reconstitutes HLA-B7 mRNA and antigen induction by Hu-IFN-gamma. Thus the segregation of cells on the basis of a biological effect by fluorescence-activated cell sorting and reconstitution by hybrid fusion provides a strategy by which some biological pathways can be mapped at a chromosomal level.

Coronary thrombolysis in a community hospital: experience with an intravenous streptokinase protocol.

To determine whether intravenous streptokinase can be delivered safely and effectively in a community hospital without acute angiography, we treated 120 patients with intravenous infusion of 1.5 million units of streptokinase shortly after arrival in the emergency room. Average time from onset of pain to treatment was 2.7 hours. Therapy was well tolerated without significant complication. Reperfusion was clinically suspected in 78% of patients. Coronary angiography was done at referral hospitals in 94% of the surviving patients two to ten days after treatment. The infarcted vessel was patent in 74% of these patients. Of these, coronary angioplasty was performed in 36%, and 32% had bypass grafting. We conclude that intravenous streptokinase can be safely and effectively used in community hospitals without acute angiography in carefully selected patients with acute myocardial infarction.

Three distinct epitopes of the human MSK20 cell surface antigen are expressed on human and nonhuman primate cells.

Five monoclonal antibodies which indirectly agglutinate human rbc were isolated and designated 2D8, 4E12, 4H10, 5A8, and 5H5. Expression of the antigen defined by 2D8 was 100% concordant with the presence of human chromosome 19 in a panel of human-Chinese hamster somatic cell hybrids. Secondary clones isolated from antigen positive hybrids exposed to antibody 2D8 and complement were shown to have lost both the cell surface antigen and chromosome 19. Small terminal deletions of 19p were observed in the karyotypes of two antigen negative clones that continued to express human GPI. All five monoclonal antibodies and the previously isolated F10 labelled the surface of parental cells and did not label the surface of deletion mutants, and the five monoclonal antibodies produced in this study inhibited the binding of F10 to the human rbc surface. Therefore, all must recognize the MSK20 cell surface antigen which was defined previously by antibody F10. Chimpanzee cells bind 5A8 and 5H5, orangutan cells bind 4E12, and neither bind 2D8 and 4H10. Therefore, the monoclonal antibodies produced in this study must recognize at least three distinct epitopes of the MSK20 cell surface antigen which are expressed on the human rbc.

Premature chromosome condensation as a predictive indicator of relapse in children and adolescents with acute leukemia: initial observations.

Premature chromosome condensation has been used to determine a proliferative potential index (PPI) in a study of children in leukemia remission at varying times during the disease. Values 35% and greater were considered predictive of relapse. Such values preceded relapse with a mean of 5 months in acute lymphoblastic leukemia (ALL) patients who had previously relapsed and in myeloid leukemia patients. ALL patients followed from diagnosis and children off therapy had fluctuating and false predictive PPI values preceding long courses of continued remission. This study suggests that the PPI as a predictive indicator for relapse may be useful for patients with ALL who have previously relapsed and for patients with myeloid leukemias. Future exploration to further evaluate this mechanism of prediction is to be attempted by investigating the ability to obtain similar and more detailed information through the use of peripheral blood rather than bone marrow samples.

Methodology of premature chromosome condensation and its potential for relapse prediction in acute leukemia of children and adolescents.

Premature chromosome condensation has been examined as a method for measuring the proliferative potential of bone marrow cells derived from children with acute leukemia with the intention of finding a predictor of relapse. A proliferative potential index (PPI) has been determined for patients with active disease at diagnosis and relapse, as well as at onset of remission and at extramedullary relapse. A modification of the technique established by Hittelman is described, which can be easily performed by the leukemia cytogeneticist. A PPI of 35% or greater is usually obtained for patients at diagnosis or in relapse. At the onset of remission, the PPI declines to values significantly below 35% and during extramedullary relapse the value of the PPI is near normal (12%). The method for the determination of the PPI is given in detail.

Analysis of human chromosome 11 by somatic cell genetics: reexamination of derivatives of human-hamster cell line J1.

Through the fusion of a CHO cell population to a human cell population, a hybrid cell line which has lost all human chromosomes except chromosome 11 was derived. This cell line, J1, does not appear to segregate human chromosome 11 during growth. A series of deletion segregants were isolated from J1 which had lost a portion of either the long, short, or both arms of chromosome 11. This panel of deletion segregants was used for mapping a number of genetic markers on the short arm of chromosome 11. Karyotypic analysis led to the interpretation that derivatives of J1 selected for the loss of cell surface antigens encoded by genes on the short arm of the chromosome had simple terminal deletions of this chromosome arm. More recently, we have applied recombinant DNA and in situ hybridization techniques to the analysis of the structure of chromosome 11. In the course of this analysis, we have obtained data that indicate that all J1 deletion segregants retain a small chromosomal segment containing the structural genes for insulin and HRAS1. Analysis of in situ hybridization data indicates that in cell lines in which a chromosome 11 fragment cannot be identified by karyotype analysis, human DNA has been translocated to a Chinese Hamster chromosome. These results suggest that the original interpretation of the karyotypes of deletion segregants derived from J1 as simple terminal deletions is not correct. A reanalysis of gene localization studies based on these deletion segregants suggests that some assignments of genes to specific bands on chromosome 11 should be reconsidered. In particular, data on additional deletion segregants are consistent with localization of the beta-globin gene complex to band 11p15. The data presented here suggest that in several hybrid derivatives of J1, a continuous DNA segment of approximately 10(7) base pairs in length which includes the insulin and HRAS1 (cellular homolog of retroviral oncogene Harvey ras) genes has been isolated from the remainder of the human genome. We propose that the stability of chromosome 11 in the original hybrid was due to complementation of a genetic defect in the original CHO cell parent by a gene located in close physical proximity to the insulin and HRAS1 genes on chromosome 11. Data are presented which test and support this hypothesis.