Roslyn E. Wallace

Characterization of WiDr: A human colon carcinoma cell line

SummaryWe describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.

Molecular and biochemical pharmacology of mitoxantrone

Evidence has been presented which indicates that Nv: intercalates DNA and additionally causes inter- and intra-strand crosslinking possibly associated with its charged side arms; there is an apparent preference for G-C base pairs; induces single strand and double strand breaks in DNA; strongly inhibits DNA and RNA synthesis; causes nuclear aberrations and chromosomal scattering; induces a block in the G2 phase of the cell cycle with an increase in cellular RNA and polyploidy; is not cell cycle phase-specific with respect to cell kill; does not induce free-radical formation; does not induce lipid peroxidation or superoxide formation; rather it may inhibit ADR-stimulated lipid peroxidation and microsomal superoxide production; does not appear to have a strong potential for cardiotoxicity on the basis of currently postulated mechanisms of action; is capable of inducing cellular resistance in vitro; resistance is associated with an apparent alteration in the cell membrane impairing drug transport into the cell. Although the precise mechanism(s) of tumor cell killing has not been fully defined it is most likely associated with an interaction by Nv with chromosomes resulting in DNA damage, which if not efficiently repaired, will lead to inhibition of nucleic acid synthesis and eventual cell death.

Development and characerization of cell lines from subhuman primates

SummarySeven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro.Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence.All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties.These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells.

Development of a diploid cell line from fetal rhesus monkey lung for virus vaccine production

SummaryThe primary goal of this study was to develop and characterize diploid cell lines from fetal tissues of subhuman primates for use in virus vaccine production. Cell lines have been established from fetal tissues of rhesus and African green monkeys, and these have been characterized according to the general criteria recommended by the International Cell Committee for Microbiological Standardization. Of these cell lines, DBS-FRhL-2, developed from lung tissue of a rhesus monkey fetus, has been found to meet the requirements of populations proposed as substrates for virus vaccines.Characterization studies show that DBS-FRhL-2 cells have a finite life of more than 50 population doublings in vitro and maintain the diploid karyotype through an active growth phase. The cells are nontumorigenic, and tests have not revealed the presence of adventitious agents. They are susceptible to a number of human viruses and can be preserved by freezing without change in virus susceptibility, cytogenetic, or growth characteristics. These results indicate the need for further evaluation of this rhesus monkey diploid cell line for acceptability as an alternate substrate in the manufacture of human virus vaccines.

Induction of alloreactive immunosuppression by 1,4-bis [(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (CL 232,468)

1,4-bis[2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione (AEAD) has been investigated for its potential immunosuppressive effect on cell-mediated immune responses. Addition of the compound to mixed lymphocyte cultures (MLC) not only significantly inhibited these cells from responding to alloantigens but also prevented the induction of cytolytic T lymphocytes (CTL). A structurally related compound, mitoxantrone, was also found to be active in inhibiting CTL induction. AEAD had to be present during the first 3 days of a 5-day MLC in order to produce a significant effect and it had no effect on those CTL already generated, suggesting that it acted upon induction of CTL rather than the effector phase. Lymphocytes from mice treated with the compound were incapable of responding to alloantigens in vitro and the effect was dose- and time-dependent. Furthermore, lymphocytes from treated mice were found to inhibit CTL generation from normal mouse lymphocytes, indicating that a suppressor cell population might be induced in the spleens of animals treated with the compound. The present findings clearly demonstrate that AEAD is a compound with potent immunosuppressive activity on alloreactive immune responses.

Development of Resistance and Characteristics of a Human Colon Carcinoma Subline Resistant to Mitoxantrone in vitro

A subline of human colon carcinoma cells (WiDr/R) resistant to the cytotoxic effects of mitoxantrone in vitro, was developed by continuous exposure to increasing concentrations of drug. After 16 culture passages in the presence of mitoxantrone, a cell population emerged which was 30-40 times more resistant to the cytolytic effect of mitoxantrone than the mitoxantrone-sensitive parent (WiDr/S) line. Resistance to mitoxantrone was retained by WiDr/R cells propagated for more than 40 cell generations in mitoxantrone-free medium. Decreased drug sensitivity was strongly associated with reduced intracellular accumulation of mitoxantrone. Moderate differences in drug retention by sensitive and resistant cells were demonstrated. However, decreased uptake due to alterations at the cell membrane which impair transport of drug into the cell, reducing interaction with DNA, appears to be the principal basis of resistance in these cells.

Changes in incidence of sex chromatin in subcultured cells.

Sex chromatin counts of subcultured cells of both female human mammary tumor and female rabbit kidney show a considerable drop from an initial high level. Cultures in which sex chromatin persists also retain the viral insensitivity of their source material.

Susceptibility of human lymphoblasts (RPMI 7466) to viral infections in vitro.

Summary Comparative viral susceptibility tests using human diploid lung fibroblasts (Led-130) and human lymphoblasts (RPMI 7466) have been made. Lymphoblasts 7466 were found resistant to the cytopathic effect of most viruses tested, but were capable of supporting persistent viral infections with many of these. Exceptions were herpes simplex and Coxsackie type A-15 viruses which were highly cytopathic for lymphoblasts 7466; viral replication in these instances was accompanied by total destruction of the culture. Concomitant virus replication and cell multiplication occurred in lymphoblasts 7466 persistently infected with poliovirus type 1 during 7 months of continuous culture. No recognizable cell destruction, reduction in growth rate, or interference to infection with two other viruses could be measured in these cultures. The mechanisms which operate to control and maintain persistent virus infections in cultures of human lymphoblasts has been studied and discussed.

Studies on Preservation by Freezing of Human Diploid Cell Strains

Summary Methods for optimal preservation by freezing of various tissue culture passages of human diploid cell strains have been investigated. Considerable loss of viability occurred when cells were slow frozen and stored at —70°C in glycerin medium. Storage loss at this temperature was reduced when DMS or a combination of glycerin and PVP was used as additive. Cell numbers per ampoule, in the concentrations tested, did not influence survival during freezing and storage at —70°C. In contrast to storage at —70°C, storage of frozen samples under liquid nitrogen refrigeration has not resulted in viability loss during a period of 1 1/2 years. Differences in viability due to the use of various additives were also smaller upon storage at these lower temperatures. Results of equilibration tests in additives prior to freezing indicate no toxicity of DMS for these cells, but prolonged exposure to glycerin was detrimental to survival. A constant cooling rate of l°C/min was not found to be critical in freeze-preservation of these cell strains, although under the conditions described, more rapid and variable cooling rates did not provide for greater cell recovery. Thawing of frozen samples in 30 or 60 seconds following storage under liquid nitrogen refrigeration gave comparable cell survival; somewhat higher viability was indicated with the faster warming rate after long term storage at —70°C. Plating efficiencies of these strains were low, but when compared in qualitative tests with pre-freeze controls, unstained cells of thawed suspensions had lost none of their ability to adhere to a plate and multiply. When returned to serial culture, recovered cells have retained full passage potential In vitro and all properties which characterize cultures of the non-frozen strains. The competent technical assistance of Mrs. Susan Kulina is gratefully acknowledged.

Arabinofuranosyl-5-azacytosine: activity against human tumors in athymic mice

SummaryArabinofuranosyl-5-azacytosine (Ara-AC), a new compound structurally related to arabinofuranosyl-cytosine (Ara-C) and 5-azacytidine (5-AC), has demonstrated significant therapeutic activity against a wide spectrum of murine tumors and three human tumor xenografts in the NCI tumor panel. Studies on the activity of Ara-AC in these and other human tumor xenograft models were undertaken to define its potential antihuman-tumor profile more completely. Ara-AC demonstrated marked antitumor activity against human tumor xenografts, including leukemias and solid tumors that do not respond to Ara-C or 5-AC. An important finding was the demonstration that Ara-AC was as effective by the oral route as when given intraperitoneally. Furthermore, the compound demonstrated synergism when combined with cisplatin in the treatment of refractory solid tumors and also induced monocyte-type differentiation of promyelocytic leukemia (HL-60) cells in vitro. Ara-AC is a promising new compound that may have utility in the treatment of human cancer beyond that anticipated for a cytotoxic nucleoside.