Chromosomal localization of the human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) gene (APAH1) to 9p13.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase is the enzyme responsible for maintaining the intracellular level of the dinucleotide Ap4A, the function of which has yet to be established. The APAH1 gene encoding this Ap4A hydrolase has been mapped by fluorescence in situ hybridization and PCR to human chromosome 9p13. Radiation hybrid panel mapping further located APAH1 between the IL11RA and the GALT genes, thus excluding it as a candidate gene for cartilage-hair hypoplasia, which maps proximal to GALT. Several tumor suppressor genes have previously been mapped within the 9p13-p21 region. Given that the FHIT gene at 3p14.2, which encodes a diadenosine 5',5"'-P1,P3-triphosphate (AP3A) hydrolase, is a candidate tumor suppressor, APAH1 should also be considered a potential tumor suppressor.

Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells.

Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.

Upregulation of O6-alkylguanine-DNA-alkyltransferase expression and the presence of double minute chromosomes in alkylating agent selected Chinese hamster cells.

Chinese hamster V79 lung fibroblasts are sensitive to the toxic effects of chloroethylating agents such as mitozolomide (Mz) and express very low levels (less than 2 fmol/mg) of the DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (ATase). These cells were subjected to selection by treatment with serially increasing doses of Mz. After each dose, the surviving population was expanded and ATase activity was determined in cell extracts. ATase specific activity increased stepwise and in cells surviving selection at 120 micrograms/ml Mz had reached 430 fmol/mg protein: polyacrylamide gel electrophoresis and fluorography showed the size of the ATase as 25 kDa. Cytological examination of these cells showed the presence of double minute (DM) chromosomes (mean approximately 3/cell) but no obvious homogeneously staining regions. In cells grown in continuous culture without further selection no marked decrease in ATase activity or DM frequency was observed. Karyotype analysis and DNA profiling confirmed that the parent and selected cells were of the same origin with, in the latter case, the probable loss or gain of a single restriction endonuclease site. No major differences were seen in the intensity of hybridization signals following Southern analyses of DNA from control and Mz selected cells using the human ATase cDNA as a probe. These results indicate that the ATase gene is not amplified in the Mz selected cells and suggest that increased ATase activity is a consequence only of increased transcription or translation of the ATase gene.

Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.

Human NK-2 receptor gene maps to chromosome region 10q11-21.

The human NK-2 receptor gene has been mapped to chromosome 10 using the polymerase chain reaction to amplify specifically the human NK-2 receptor sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids. The assignment to chromosome 10 was confirmed by in situ hybridisation to human metaphase chromosomes, giving a regional localisation of 10q11-21.

Detection of mRNA for ribosomal phosphoprotein P2 in normal colon and colonic tumours by in situ hybridization.

We have localized the mRNA for the ribosomal phosphoprotein P2, a putative metastasis-related sequence, in normal colon and colonic carcinomas by in situ hybridization, using an oligonucleotide probe end-labelled with digoxigenin. The mRNA was identified in normal colonic epithelial cells, the intensity of the signal being greater in cells at the base of the crypts compared with those on the surface. A strong positive signal was also seen in plasma cells, in fibroblasts in granulation tissue, in ganglion cells, and in hepatocytes. A positive signal was identified in all 16 primary colonic tumours studied and in 7 hepatic metastases. In contrast to previous studies based on Northern blot analysis, we were unable to demonstrate increased expression in metastases as compared with primary tumours, nor could we demonstrate any increased expression in primary tumours which were associated with distant metastases.

The human aldose reductase gene maps to chromosome region 7q35.

The human aldose reductase (AR) gene has been mapped to chromosome 7 using the polymerase chain reaction to specifically amplify the human AR sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids. The assignment to chromosome 7 was confirmed by in situ hybridisation to human metaphase chromosomes using a novel, rapid hybridisation, method giving a regional localisation at 7q35.

Trophoblast glycoprotein recognised by monoclonal antibody 5T4 maps to human chromosome 6q14-q15.

Human X rodent hybrids were stained by indirect immunofluorescence with 5T4, a murine monoclonal antibody that recognises a 72 kdalton glycoprotein expressed by human trophoblasts and a very restricted range of adult tissues; they were analysed by flow cytometry. Concordance analysis supported by segregation data allowed assignment of the gene controlling glycoprotein expression (M6P1) to chromosome 6. Similar analysis with translocation hybrids gave a regional assignment to 6q14-q15. M6P1 is distinct from NT5, coding for 5' nucleotidase, which maps to the same region.

Expression of human CD4 by two human-mouse interlineage hybrids.

Two hybrid cell lines expressing human CD4 were prepared by fusing human B-lymphoid cells with the mouse T-lymphoma BW5147. Hybrid TF42 was derived from a human B-lymphoblastoid line and TF53.1 from a human B-ALL. Variants of these hybrids expressing or lacking CD4 were isolated by sorting cells stained with the monoclonal antibody (mAb) OKT4 on a fluorescence-activated cell sorter (FACS). Cytogenetic, isoenzyme and DNA analysis confirmed the presence of human chromosome 12 in the CD4+ hybrids, and revealed that CD4 expression by TF42 was associated with multiple copies of this chromosome. Of seventy mAb recognizing human T-cell antigens screened on the CD4+ and CD4- variants of the two hybrids, only mAb recognizing CD4 and Leu 8 reacted with the CD4+ cells. These hybrids should be useful in the preparation, screening and analysis of anti-CD4 monoclonal antibodies, and in studies of CD4 epitopes recognized by HIV.

Increased O6-alkylguanine alkyltransferase activity in Chinese hamster V79 cells following selection with chloroethylating agents.

Chinese hamster V79 lung fibroblasts express low levels (specific activity 2-4 fmol/mg protein) of O6-alkylguanine (O6-AG) alkyltransferase (ATase). In cells surviving selection with low doses (10 micrograms/ml) of the chloroethylating agent, mitozolomide (Mz), ATase activity was increased 5- to 8-fold. Repeated selection of such cells produced a maximal specific activity of 36-40 fmol/mg protein, whilst selection at 20 or 40 micrograms/ml result in specific activities of approximately 50 and 70 fmol/mg respectively. Only slight decreases in ATase activity were seen by 51 days after an initial selection with 10 micrograms/ml Mz. A similar effect was observed using chlorozotocin. Selected cells had a higher D37 for Mz (2.5-6.0 micrograms/ml) in comparison with control cell (D37, 0.8 micrograms/ml) but the D37s for nitrogen mustard and vincristine were closely similar in selected and control cells. Possible explanations for the increase in ATase activity are discussed.

The c-Harvey-ras-1 oncogene in chromosome mediated gene transfer.

Transfection of DNA derived from a variety of tumours can induce morphological transformation of certain immortalised but normally contact-inhibited cell lines. This important technique has been instrumental in the identification and subsequent molecular cloning of a number of oncogenes, including Harvey-ras. We can extend this approach for studying neoplastic potential by performing chromosome-mediated, as distinct from DNA-mediated, gene transfer. This modification offers two potentially important advantages, both of which stem from the fact that sub-chromosomal lengths of DNA are transferred. Firstly, the expression of the oncogene can be studied in its normal chromosomal milieu; potential modifying effects of linked and unlinked sequences can be evaluated. Secondly, new chromatin segments with transforming potential but too large to be transferred as naked DNA may be revealed. Our experiments illustrate some of the new insights into the molecular basis of neoplastic change which can be gained by this technique. They also demonstrate the power of the technique as a genetic tool for the isolation and detailed molecular analysis of oncogene-associated, sub-chromosomal regions of the human genome.

HRAS1-selected, chromosome-mediated transformants vary in phenotype in vitro and tumorigenic potential in vivo.

Transfection of mouse C127 cells with mitotic chromosomes isolated from a human EJ bladder carcinoma cell line gave rise, at high frequency, to foci of transformed cells. Independent, HRAS1-selected chromosome-mediated transformants displayed distinctive cellular morphologies in monolayer culture and colony-forming abilities in low-melting-point agarose. Subcutaneous inoculation of neonatally thymectomized, Ara-C-protected, total-body-irradiated CBA mice was used to compare the tumorigenic potential of each transformant. Significant quantitative and qualitative differences in tumorigenicity were found between transformants which correlated with differences in malignant phenotype observed in vitro. The sensitivity of the tumorigenicity assay is such that rare transformation events can be selected directly in vivo.

Gene mapping and physical arrangements of human chromatin in transformed, hybrid cells: fluorescent and autoradiographic in situ hybridization compared.

We compare a fluorescent in situ hybridization technique, using N-acetoxy-2-acetylaminofluorene (N-ACO-AAF) modified DNA adducts, with 3H-labeled DNA in situ hybridization for visualizing human transgenomes in HRAS1-selected, chromosome-mediated gene transfer (CMGT), and mapping chromosomal SV40 in an SV40-transformed, human-mouse hybrid cell line. We demonstrate that individual HRAS1-CMGTs may contain multiple fragments of human chromatin. We deduce that the CMGT process can involve interstitial loss of mouse chromatin. We conclude that the N-ACO-AAF technique gives finer resolution than 3H-labeled in situ hybridization. However, 3H-labeling is more sensitive and has allowed us to sublocalize SV40 in C121 to the region 7q31-35.

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.

The gross pathology and histological features of tumours produced by inoculation of human cell lines into immune-deprived mice.

Tumours were raised in both congenitally athymic ('nude') Swiss mice and in neonatally thymectomized, Ara-C-protected, whole-body irradiated CBA mice by subcutaneous inoculation of cells from a variety of cultured human lines. In both types of animal, tumours tended to grow massively at the site of inoculation, with some infiltration of adjacent tissues but only rarely with evidence of metastatic spread. Tumours derived from Burkitt's lymphoma (BL) lines or from EB virus-transformed lymphoblastoid cell lines (LCL) were all classified as high grade malignant lymphomas with a limited range of appearances on conventional histological examination. In the material studied there were no consistent features distinguishing BL-derived from LCL-derived tumours. Cell lines originating from other haematopoietic malignancies tended to produce tumours interpreted as immunoblastic lymphomas though there were distinctive characteristics in some cases, such as highly convoluted or pleomorphic nuclei in the cells of some tumours derived from T-cell leukaemia lines and plasmacytoid differentiation in tumours originating from myeloma lines. Malignant cell lines of epithelial origin gave rise to tumours with the histological appearances of anaplastic carcinomas readily distinguishable from the high grade lymphomas produced by haematopoietic cells.

Cellular studies on retinoblastoma.

Retinoblastoma may be hereditary or non-hereditary. The hereditary form involves either a predisposing gene transmissible as an autosomal dominant or a deletion at chromosome 13q14. An abnormal cellular response to ionizing radiation was suggested by the occurrence of secondary neoplasms within the field of therapeutic radiation in hereditary retinoblastoma patients. Hereditary retinoblastoma patients also show a predisposition to second neoplasms not related to therapy. In vitro studies on the radiation response of cells from retinoblastoma patients have generated conflicting results. Some laboratories, including our own, find that survival following ionizing irradiation of fibroblasts is within the normal range, other laboratories find an abnormal decrease in cell survival. X-ray-induced chromosome damage in G0-irradiated lymphocytes was slightly elevated compared to control subjects. Recent studies using chromosome 13 genetic markers suggest that retinoblastoma tumour cells are homo- or hemi-zygous for the mutant retinoblastoma gene. It seems unlikely that the mutant gene causes sensitivity to ionizing radiation but any tendency to chromosomal rearrangement in a gene carrier would increase the probability of tumour development.

The suitability of immunosuppressed mice kept in a standard animal unit as recipients of human tumour xenografts.

CBA/Lac mice were immunosuppressed by thymectomy and whole body irradiation with 250 kVp X-rays following pretreatment with cytosine arabinoside. The optimum radiation dose for immunosuppression with prolonged survival was 7.35 Gy. The animals were kept in a standard animal unit with an overall survival rate of 83%. They were found to be suitable for large scale, long-term, xenotransplantation experiments at 20% of the cost of nude mice.

The cytogenetics of human B lymphoid malignancy: studies in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid cell lines.

Cells from Burkitt's lymphoma (BL) and from the majority of human B-cell neoplasms show karyotypic changes that characteristically involve chromosomal breakage and recombination in addition to some chromosome gains. These aberrations increase as the tumours progress in vivo, and a similar tendency is seen in BL-derived lymphoid lines in vitro. Epstein-Barr virus (EBV)-transformed lymphoblastoid lines of non-malignant origin also develop karyotypic abnormalities on prolonged culture, but these are predominantly nonrandom gains of whole chromosomes (i.e., non-disjunction events). They have never been observed to acquire the 8;14 translocation, which is an almost constant feature of BL. Nevertheless, there is some concordance between the pattern of chromosome gains found in long-term cultured lymphoblastoid lines and that seen in direct preparations from B-cell neoplasms. Many of the lymphoblastoid lines that have become aneuploid are tumorigenic in immunosuppressed mice, indicating that EBV-transformed human B cells can acquire a malignant phenotype in the absence of specific chromosomal translocations. It is suggested that the predominance of chromosomal breakage and recombination events in the karyotypic evolution of BL and other lymphoid neoplasms comes about because chromosomal instability (which varies within a population) is a major risk factor for lymphoid malignancy, interacting with other risk factors, including impaired T-cell function and EBV to determine the clinical and epidemiological patterns of BI and related neoplasms.

Tumorigenicity of human lymphoblastoid cell lines, acquired during in vitro culture and associated with chromosome gains.

Tumorigenicity of human lymphoma and lymphoblastoid B-cell lines was assessed by their ability to form growing and transplantable masses on subcutaneous inoculation into neonatally thymectomized, Ara-C-protected, total-body-irradiated mice. By these criteria, 12 lines of known malignant origin were tumorigenic, 11 lymphoblastoid lines, tested after less than one year of in vitro growth, were non-tumorigenic and 8/18 long-established lymphoblastoid lines produced transplantable tumours. All of the long-established lines had acquired karyotypic changes on prolonged culture, the predominant characteristic being a gain of whole chromosomes or of major chromosome segments. None showed the classical 8:14 translocation associated with Burkitt's lymphoma. Comparisons with nontumorigenic precursors (recovered from liquid nitrogen storage) and with other non-tumorigenic but chromosomally abnormal, lymphoblastoid lines suggest that imbalance of the dosage of genes carried on chromosomes 7,8 and 9 may be important in determining the tumorigenic phenotype.