Inter- and intraspecies contamination of human breast tumor cell lines HBC and BrCa5 and other cell cultures.

It is shown that the two most recently reported cell lines derived from malignant human breast tissue, HBC and BrCa5 are, respectively, rat and HeLa cell contaminants. The incidence of inter- and intraspecies contamination among 279 cell cultures from 45 laboratories in an 18-month survey is also presented.

HeLa cultures defined.

A list is presented of references to all known publications on properties which have served to relate strains of HeLa cells to each other as well as to indict other purported human cell lines as HeLa cell contaminants. Eleven additional cell lines not previously indicted are described. When they exhibit (i) type A (fast) mobility for glucose-6-phosphate dehydrogenase, (ii) phosphoglucomutase type 1 at locus 1 and locus 3, (iii) absence of a Y chromosome by fluorescent staining, and (iv) possession of a complex of trypsin-Giemsa banded marker chromosomes present in known HeLa cells, then cell substrates regardless of designation should be considered de facto strains of HeLa.

Banded marker chromosomes as indicators of intraspecies cellular contamination.

Chromosome banding revealed marked chromosomes characteristic of HeLa cells in cultures designated HEK, HEK/HRV, HBT-3, HBT-39B, MA160, and a strain of SA-4TxS-Husa(1). Ohter HeLa cell characteristics found were glucose-6-phosphate dehydrogense type A mobility and lack the Y chromosome. Conventional chromosome analysis and immunological and enzymatic technique serve to monitor species specificity and racial origin of the donor. Chromosome banding, however, can monitor intralinear karyotype peculiarity and its evolution during long-term cultivation.

Cross-contamination of cells in culture.

Lists are presented of references to all known publications describing cell properties that serve to characterize (i) known strains of HeLa and purported human cell lines indicated as HeLa contaminants, (ii) strains of human cell lines contaminated with human but non-HeLa cells, and (iii) strains of cells contaminated by cells from one or more other species. Frequencies of cell cross-contaminations are cited and references are presented to relatively simple techniques that could serve to detect such contamination.

Prenatal in situ hybridization test for deleted steroid sulfatase gene.

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specifically to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome-specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders.

Prenatal diagnosis with repetitive in situ hybridization probes.

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY, +21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries.

Distinctive banded marker chromosomes of human tumor cell lines.

Nine human tumor cell lines (five breast carcinomas and four sarcomas) have been studied and each revealed groups of distinctive banded marker chromosomes which can serve to identify them and aid in monitoring cell line specificity. This was possible neither by conventional karyology in terms of numbers and morphology of chromosomes nor by glucose-6-phosphate-dehydrogenase mobility which was type B for all cultures. The significance of the clonal nature of the cell lines is discussed.

Human embryonic lung cells (HEL-R66) are of monkey origin.

Two cultures of cells designated HEL-R66, presumable of human origin, revealed monkey instead of human cell characteristics, including chromosomes, isoenzyme mobility pattern and species-specific cell membrane antigen.

Specific chromosome loss associated with the expression of tumorigenicity in human cell hybrids.

Four related nontumorigenic and tumorigenic HeLa x fibroblast intraspecific human hybrid cell lines were analyzed to determine whether specific chromosome(s) are associated with the control of tumorigenic expression. The loss of one copy each of both chromosome 11 and chromosome 14 were associated, with a high degree of statistical significance, with the expression of tumorigenicity in two segregants derived from the original nontumorigenic hybrid population. Although the parental origin of the chromosomes could not be established in this study, our preliminary results suggest that complex, genetically determined, regulatory interactions may operate in the control of neoplastic expression.

Rapid aneuploid diagnosis of high-risk fetuses by fluorescence in situ hybridization.

OBJECTIVE: Our purpose was to develop fluorescence in situ hybridization to repetitive chromosome-specific sequences to detect chromosome aneuploidy faster than hybridization to unique targets or karyotyping. STUDY DESIGN: Aneuploidy involving chromosomes 13, 18, 21, X, and Y comprises 70% of chromosome abnormalities in 10- to 12-week fetuses, 95% of the phenotypically significant newborn chromosome abnormalities. Our improved 8-hour protocol used repetitive probes to label and count the number of these centromeric chromosome domains. RESULTS: This protocol correctly determined chromosome 13, 18, and 21 status in 50 of 50 unselected direct amniocyte samples and found abnormal patterns in 27 of 27 archived trisomy 21 cases. Altogether karyotyping confirmed 744 of 745 chromosome-specific repetitive sequence test results. CONCLUSION: This protocol rapidly tests abnormal fetuses and newborn infants in whom diagnosis is made at the initiation of labor or before urgent surgery when a cytogenetic result cannot be completed.

Human tissue factor: cDNA sequence and chromosome localization of the gene.

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)