A protein from Naegleria amoebae causes apoptosis in chick embryo and CHO cells after they become confluent.

Exposure for less than an hour to a protein isolated from Naegleria amoebae initiates a process that has no apparent effect on the appearance or growth of chick embryo or CHO cell cultures for 4 to 9 days; after the development of confluency, at some unknown signal, all of the cells undergo an apoptotic death within a 12- to 24-hour period. Abnormalities detected among the last mitotic cells include chromosomal breakage and early reversal in metaphase to telo/interphase daughter nuclei with irregular shapes. Additional events in the dying cultures include the development of a cytoplasmic amoebic-related immunogen, gross DNA fragmentation, cell blebbing, shrinkage, and apoptotic body formation. Culture death included all cells, those present in confluent cultures when the protein was added, and in other cultures, those formed during a more than 30-fold increase in cells as the cultures became confluent. The increase in the number of cells followed by the uniformity and synchrony of their death pattern indicates that the signal to kill has increased and spread throughout the culture; upon an unknown condition related to confluency, events are initiated that lead to the unusual apoptotic death of the culture.

Chromosome analyses of human mammary epithelial cells at stages of chemical-induced transformation progression to immortality.

Benzo(a)pyrene induced extended life (EL) (i.e., a longer than normal proliferative lifespan before senescence) of human breast cells in culture. From many EL cell cultures immortalized cells emerged only once in each of two separate experiments. The original EL cells were mostly normal diploid with only a small percentage of tetraploid cells. The two immortalized cell lines, however, were near diploid, each containing a set of chromosomal aberrations that were present in all the cells analyzed, confirming the clonal origin of both cell lines. For cell line 184A1 the aberrations consisted of deficiencies only, whereas a combination of deficiencies and duplications characterized the 184B5 line. None of the individual aberrations of each set were shared by both cell lines. Both sets of aberrations have remained stable for over 150 population doublings, while some of the other chromosomes showed breakage and reunions. These data are discussed in regard to types of mutations in the sequence of changes from primary to immortalized cells, and it is concluded that the sets of aberrations most likely originated as multiple events in a single cell.

Transformation of human mammary epithelial cells by oncogenic retroviruses.

We have introduced viral oncogenes into human mammary epithelial cells through the use of murine retroviruses. A continuous cell line (184A1N4) derived from benzo(a)pyrene treatment of normal breast epithelial cells was used as a recipient for the ras, mos, and T-antigen oncogenes. Each of these oncogenes enabled the 184A1N4 cells to grow in a selective medium, thus demonstrating the potential utility of these cells for oncogene detection and isolation. 184A1N4 cells transformed by T-antigen were nontumorigenic in athymic mice, but v-ras transformants were weakly tumorigenic. Transformants bearing both the T-antigen and ras oncogenes were strongly tumorigenic, however. The karyotype of these double transformants shows a high degree of stability. These results demonstrate the stepwise acquisition of the fully malignant phenotype by normal human epithelial cells in vitro.

Coxsackievirus B-3 selection of virus resistant Buffalo green monkey kidney cells and chromosome analysis of parental and resistant cells.

Following coxsackievirus B-3 (CBV-3) infection and lysis of highly susceptible Buffalo green monkey kidney (BGMK) cells, there was a regrowth of cells. Cultures of regrown cells were established and they continued to release infectious CBV-3 for up to 20 weeks. The parental BGMK cells were susceptible to CBV-3, CBV-4 and poliovirus type 2 induced cytopathic effect (CPE), while the cured cells were resistant to CBV-3 and CBV-4 but not to poliovirus type 2. Attachment of CBV-3 was restricted on cured BGMK cells but not on parental cells. Chromosome analysis showed that the cured cells originated from the BGMK cell line and that they were missing two marker chromosomes present in the parental cells.

Chromosome instability in cell lineages of amniocyte clones morphologically transformed by Simian virus 40.

Seven clonally derived human amniocyte cell lines from independent Simian virus-40 transformation events were analyzed for chromosomal aberrations in the precrisis stage. The distribution of chromosomal aberrations over the haploid complement was nonrandom. With increasing passage number the number of involved chromosomes increased, but the type of aberrations were the same: dicentric chromosomes and translocations due, respectively, to fusion of telomeres and breakage in the centromere regions. Interruptions to telomeres were also evident from extrachromosomal material terminally on the p arms, especially, of chromosomes #7 and #11. The cytogenetic similarities between the present results and those in transfection experiments are mentioned. All seven clones became highly heterogeneous over 10 to 20 passages (i.e., 30-60 population doublings) when, in general, transformed amniocytes enter crisis. The chromosomes which were nonrandomly involved in aberrations exhibited great diversity in types of rearrangements. Thus, for each clone multiple different rearrangements for specific chromosomes were observed in different cells. Most of these aberrations had a single step origin, which implies that breakage occurred repeatedly for the same chromosome in different cells, suggesting that the specific chromosome had become unstable in an earlier cell generation. Thus, we observed development of clonal chromosome instability, a phenomenon not reported for viral transformed cells.

Induction of tumorigenesis and chromosomal abnormalities in human amniocytes infected with simian virus 40 and Kirsten sarcoma virus.

Cell cultures of epithelial-like human amniocytes were infected with simian virus 40 (SV40) and Kirsten sarcoma virus (KSV) in various sequential orders, and tested for agar growth, chromosome abnormalities, and tumorigenesis in the nude mouse assay. We observed that regardless of the order in which the viruses were introduced, the doubly infected cells always exhibited the typical SV40 premalignantly transformed phenotype before changing to the malignant phenotype. All doubly transformed cells from different cell donors produced tumors in adult and suckling nude athymic mice, classified as poorly differentiated sarcomas. Infection with SV40 alone conferred extended life span and accelerated growth without the malignant capability of tumor production. Kirsten sarcoma virus alone produced only focal cell alterations with no change in cell longevity or tumorigenesis. Chromosome studies of the premalignant and malignant cells from one cell donor did not reveal any significant clonal development for marker chromosomes in either cell line. Chromosome 12, which carries the homologous cellular oncogene to KSV, had no increase in aberrations in the malignant cells. Chromosome 8 was most often involved in aberrations, and the most frequent aberration for both series was dicentric chromosomes due to telomere fusion. For other translocations the breakpoints were almost exclusively in the centromere regions. The vulnerability of telomere and centromere regions to the free virus present in these precrisis cells is discussed, and similarities in regard to types of aberrations in transfection experiments are noted.

Anchorage independent growth of SV40 transformed human epithelial cells from amniotic fluids: differences within and among cell donors.

Cells from seven individuals were cultured separately for the induction of morphological transformation by SV40. Sixty-three transformed colonies were tested for anchorage independent growth in soft agar at various passage levels. Colony formation was consistent for all clones of respective cell donors. Four donors yielded clones that grew in soft agar in the first passage. Clones from three donors were similar to controls and formed no colonies. The size of the agar colonies was constant in the early passages. Size differences were observed in later passages and for negative clones that gained anchorage independence during time in culture. The early passage positive type of anchorage independence is expressed concomitantly with morphological transformation. Considering that the clonal isolates were genetically homogeneous within cell donors and heterogeneous among cell donors, it is concluded that the phenotype of anchorage independence is determined by at least two genetic mechanisms; namely, the genotype of the cell donor (the hereditary type) or by culturally derived new genetic variability, or both. Family history on cancer incidence showed that one grandparent for each of the four positive donors for the hereditary type of anchorage independence had cancer, whereas the grandparents of the three negative donors were asymptomatic. The incidence of cancer did not appear to be age related. Chromosome analyses of two morphologically transformed colonies from each of the cell donors by the in situ technique, showed diploid and tetraploid cells and a small number of cells with rearrangements. It is concluded as previously that the progenitor transformed cell to the colony of cells is normal diploid.

Improved immunofluorescence antigens for detection of immunoglobulin M antibodies to Epstein-Barr viral capsid antigen and antibodies to Epstein-Barr virus nuclear antigen.

Epstein-Barr viral capsid antigen and nuclear antigen produced by modified procedures were evaluated for use in measuring viral capsid antigen immunoglobulin M and Epstein-Barr virus nuclear antigen antibody responses in sera from patients with suspected Epstein-Barr virus infections. Viral capsid antigen production was stimulated with a phorbol ester, and the Epstein-Barr virus nuclear antigen cells were fixed in suspension to eliminate loss of antigen during the drying process. Both preparations proved to be sensitive and reliable.

Chromosome analyses and anchorage-independent growth of SV40-induced morphologically transformed epithelial cells from amniotic fluids.

Epithelial cells from amniotic fluid cell cultures are morphologically transformed by simian virus 40, 20 to 30 d after infection. The cells of the transformed colonies are highly basophilic, have a high nuclear-to-cytoplasmic ratio, and show a dense growth pattern. The cells are virus producers, and ultimately, after continuous passage, the cell lines reach a crisis situation with no growth. Twelve morphologically transformed cell colonies were isolated from five different individuals for chromosome analyses after approximately 18 population doublings (second bottle passage). For all cell lines diploid cells were observed. Banding of the chromosomes revealed normal morphology of euchromatic and heterochromatic regions. The suggestion is made that chromosome alteration is not necessary, nor a prerequisite, for the morphologically transformed phenotype to be expressed and that the transformation process per se causes chromosomal instability. Tests for colony formation of the 12 cell lines in semisolid medium showed that different transformed colony isolates from the same individual donor of the cells either formed or did not form colonies in agar. The size of the colonies was also consistent within individuals as compared to between individuals. These limited results are suggestive of a dependence upon the genetic constitution of the individual donor of the cells for colony formation in soft agar.

Nuclear involvement poin xvirus infection.

During the first hours of infection with vaccinia virus, part of the chromosomal DNA of host cells is degraded, part of it is released from the cell, and part of it eventually appears in cytoplasmic foci associated with the reproduction of virus. The DNA of the infecting particle is first found attached to the chromosomes. Intranuclear foci of DNA incorporation appear later; these rapidly move out into the perinuclear space and eventually become typical cytoplasmic "virus factories." Each of these observations points to a more direct involvement of the nucleus in viral infection than was previously envisaged.