2-Chloroethyl-3-sarcosinamide-1-nitrosourea, a novel chloroethylnitrosourea analogue with enhanced antitumor activity against human glioma xenografts.

Nitrosoureas are among the most widely used agents used in the treatment of malignant gliomas. Here, the activity of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) was compared with that of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), in vivo against s.c. implanted SF-295 and U-251 central nervous system (CNS) tumor xenografts. When given i.v., q4d for 3 doses, to athymic mice bearing s.c. SF-295 tumors, SarCNU, at an optimum of 167 mg/kg/dose, produced 9 tumor-free animals of 10 total animals, 1 regression, and no evidence of overt toxicity (> or =20% body weight loss). With a similar dosing schedule, BCNU produced no tumor-free animals, six regressions, and one drug-related death at its optimum of 30 mg/kg/dose. Furthermore, SarCNU retained high antitumor activity at two lower dose levels, 66 and 45% of the optimal dose, whereas BCNU demonstrated a progressive loss of antitumor activity at lower doses. Following p.o. administration, SarCNU similarly demonstrated antitumor activity that was superior to that of BCNU. In the U-251 CNS tumor model, SarCNU yielded six of six tumor-free animals at 80 mg/kg/dose with i.p. administration q.d. for 5 days, starting on day 14, whereas BCNU, at 9 mg/kg/dose, yielded three of six tumor-free mice and one drug-related death. Again, SarCNU resulted in tumor-free animals at 66 and 45% of its optimal dose and was relatively nontoxic, in contrast to BCNU. Results of testing to date indicate that SarCNU is clearly more effective than BCNU against the human CNS tumors SF-295 and U-251 in vivo. These results encourage the initiation of clinical trials for SarCNU, in an effort to improve therapeutic approaches to glioma, but clinical trials must determine whether superiority of SarCNU in preclinical models can be extrapolated to patients.

Reductase enzyme expression across the National Cancer Institute Tumor cell line panel: correlation with sensitivity to mitomycin C and EO9.

BACKGROUND: Many antitumor drugs require metabolic activation to exert their cytotoxic or cytostatic effects. The so-called bioreductive compounds, whose conversion into active antitumor agents is catalyzed by reductase enzymes, are examples of such drugs. The identification of specific enzymes involved in the activation of these compounds is important in understanding cellular factors that may influence drug antitumor activity. PURPOSE: We measured expression levels of three different reductase enzymes-DT-diaphorase [NAD(P)H (i.e., reduced nicotinamide adenine dinucleotide, with or without phosphate): quinone oxidoreductase]; NADPH:cytochrome P-450 reductase; and NADH (i.e., reduced nicotinamide adenine dinucleotide): cytochrome-b5 reductase- in 69 cell lines (most of the National Cancer Institute [NCI] human tumor cell panel) to see if relationships could be established between the activities of these enzymes and cellular sensitivities to the bioreductive compounds mitomycin C and EO9. METHODS: For all 69 cell lines, the activity of each enzyme was determined using cellular extracts and photometric assays involving the reduction of cytochrome c. Western blot analysis was used to measure the relative amount of DT-diaphorase protein in each extract, and coupled reverse transcription and polymerase chain reactions were employed to assess DT-diaphorase and NADPH:cytochrome P-450 reductase messenger RNA (mRNA) levels in a subset of the cell lines. The cytotoxic and/or cytostatic activities of mitomycin C and EO9 toward the cell lines were determined under aerobic conditions. Relationships between enzyme activity levels and drug sensitivities were assessed by use of the COMPARE program and Pearson correlation coefficients. RESULTS: In general, DT-diaphorase activity levels were higher than those observed for the other two reductases across the entire cell line panel. Measured activities for all three enzymes varied among cell lines derived from the same tissue as well as between lines derived from different tissues; however, tissue-specific patterns of expression could be discerned. Differences in the activity levels of individual enzymes appeared to reflect differences in corresponding enzyme protein and/or mRNA levels. A relationship between enzyme activity and chemosensitivities to mitomycin C and EO9 was observed only for DT-diaphorase (Pearson correlation coefficient = .424 [two-sided P<.0005] for mitomycin C and .446 [two-sided P< or = to .0013] for EO9). CONCLUSIONS: Reductase enzyme expression is heterogeneous across human tumor cell lines, and tissue-specific patterns of expression are apparent. DT-diaphorase activity levels correlate with sensitivities to mitomycin C and EO9, supporting a role for this enzyme in the bioactivation of these anticancer compounds. IMPLICATIONS: Comparison of biochemical, molecular biological, and chemosensitivity data obtained from screening a large number of cell lines (e.g., the NCI tumor cell line panel) may facilitate investigation of factors influencing drug antitumor activity. The knowledge gained may be of value in the development of new anticancer agents or in the selection of patients to receive specific therapies.

In vivo cultivation of tumor cells in hollow fibers.

Advancement of potential anti-cancer agents from "discovery" in an in vitro screen to pre-clinical development requires a demonstration of in vivo efficacy in one or more animal models of neoplastic disease. Most such models require considerable materials in terms of laboratory animals and test compound as well as substantial amounts of time (and cost) to determine whether a given experimental agent or series of agents have even minimal anti-tumor activity. The present study was initiated to assess the feasibility of employing an alternate methodology for preliminary in vivo evaluations of therapeutic efficacy. Results of experimentation to date demonstrate that a hollow fiber encapsulation/implantation methodology provides quantitative indices of drug efficacy with minimum expenditures of time and materials. Following further pharmacologic calibrations, the hollow fiber technique is anticipated (a) to identify compounds having moderate to prominent anti-cancer activity and (b) to facilitate the identification of sensitive tumor cell line "targets" and optimal or near-optimal treatment regimens for subsequent testing using standard in vivo solid tumor models. The potential suitability of this methodology is demonstrated with several standard anti-neoplastic agents.

Feasibility of cellular microencapsulation technology for evaluation of anti-human immunodeficiency virus drugs in vivo.

We investigated the feasibility of micro-encapsulation technology for the evaluation of anti-human immunodeficiency virus (HIV) drugs in vivo. The ability to place human cells in microcapsules with semipermeable membranes for implantation into test animals led to the development of this assay. The anti-HIV activity assay involves microencapsulating human T-lymphoblastoid cells sensitive to the cytopathic effects of HIV; the encapsulated cells are then implanted into athymic nude mice and recovered after drug treatment in vivo. A positive antiviral effect of the test substance is indicated by growth or survival of the virus-infected cells in the microcapsules. Several HIV-sensitive cell lines of T-lymphocyte, monocyte, and nonlymphocyte origin were examined for growth in microcapsules in vitro and in vivo. Light and electron microscopic analysis of the capsules and the human cells contained therein revealed the invasion of mouse immune cells and other adverse effects that could not be overcome by any of numerous technical modifications attempted. We conclude that cellular microencapsulation technology is not feasible for in vivo drug-testing protocols because of immunogenic reactions.

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium.

Mouse keratinocytes cultures readily develop into established cell lines without undergoing a "crisis" in a newly-developed serum-free medium, LEP/MK2. LEP/MK2 consists of calcium-free MEM with non-essential amino acids supplemented with 8 factors. Two lines, MK1 and MKDC4, have been isolated and have now doubled more than 400 and 200 times respectively. In MK1 cells, Giemsa banding has revealed significant karyotypic changes as early as the 4th passage, leading to a near-tetraploid karyotype with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers have been observed. After these initial changes, examination of cultures at several passage levels has shown that the karyotype has remained essentially stable. The MKDC4 line, also sub-tetraploid at the 7th passage, had 4 marker chromosomes by the 47th passage. The rapid increase in chromosome number may have contributed to the "immortalization" of these lines. The response of these established keratinocyte lines to growth factors and serum-derived inhibitors changed with increasing passage level. Most notable of these changes were a reduction in the requirement for bovine pituitary extract (an absolute requirement for growth of secondary MK1 cells) and a decreased sensitivity to serum and serum-derived inhibitors, e.g., transforming growth factor-beta. The established lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal differentiation and are non-tumorigenic in athymic, nude mice. This serum-free system is suitable for transformation studies with oncogenes and chemical carcinogens.

Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes.

Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64-80, 1984). This serum-free system was used to investigate the activity of fetal bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca2+ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major alpha-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-beta) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes.

Repair of DNA-protein cross-links in an excision repair-deficient human cell line and its simian virus 40-transformed derivative.

DNA-protein cross-links are induced in mammalian cells by X-rays, ultraviolet light, fluorescent light, and numerous chemical carcinogens. Others have shown that these cross-links are repaired by normal cells but that excision repair-deficient xeroderma pigmentosum (XP) Group A cells, XP12BE, are deficient in repair of these bulky adducts. This paper compares the DNA-protein cross-link repair competency of another XP Group A strain, XP20S, with its more rapidly proliferating simian virus 40-transformed derivative line and with normal human skin fibroblasts. DNA-protein cross-links were induced with 20 microM transplatinum(II)diamminedichloride and assayed by the membrane alkaline elution procedure of Kohn. Treated and untreated cells are lysed on a polycarbonate membrane filter, and the coelution rates of the DNA at pH 12.2 are compared; DNA-protein cross-links retard elution of DNA. The repair competency of XP20S cells for trans-platinum(II)diamminedichloride-induced DNA-protein cross-links was similar to that of XP12BE cells, but the competency of the simian virus 40-transformed XP20S cells was nearly equal to that of normal human skin fibroblasts. These results suggest that either cell cycling compensates for the genetic deficiency present in the nucleotide excision process of XP Group A cells or that a process other than nucleotide excision can repair these lesions; this process requires cell cycling or activation by the virus.

Approaches to enhance proliferation of human epidermal keratinocytes in mass culture.

Because of interest in mechanisms of carcinogenesis in human epithelial cells, quantitative procedures were developed for the mass culture of human epidermal keratinocytes in the absence of feeder cells. Several approaches were used to enhance proliferation since target cells are considered most susceptible to transformation if they are treated with carcinogenic agents during DNA synthesis. Mass cultures of enzymatically dispersed human foreskin were initiated in collagen-coated flasks containing medium NCTC 168 with 10% Chelex 100-treated horse serum. Under these conditions, human keratinocytes required a higher calcium ion concentration ([Ca2+]) than that reported for suspensions plated at low cell density. Neither the cohesiveness of the epidermal sheet nor continued proliferation was maintained by 0.15 mM Ca2+; 0.3 mM Ca2+ maintained these properties in primary culture only. A concentration of 1.0 mM Ca2+ provided the highest cell yield for prolonged growth as determined by the enumeration of cell nuclei isolated by citric acid. Reproducibility of successful initiation was achieved by inoculation of cells into a medium designed for clonal growth followed by culture in medium NCTC 168. Thus the balance of nutrients and electrolytes must be adjusted to satisfy the requirements of a dynamically expanding keratinocyte population.

Modulation of epithelial cell proliferation in culture by dissolved oxygen.

Modulation of epithelial cell proliferation by the dissolved oxygen concentration (PO2) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epithelial cell line (LLC-MK2). Direct measurement of the growth medium PO2 provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2. Sustained proliferation of LLC-MK2 cells occurs in serum-free medium equilibrated with a gas phase containing 18% or 30% O2 v/v. Mid-logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60-70 mm Hg decline in PO2, and leads to a stable growth medium PO2 between 70 and 100 mm Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gass phase containing 0% or 1% O2 v/v to yield a growth medium PO2 - approximately 20-40 mm Hg, proliferation of LLC-MK2 and primary foreskin epithelial cells is retarded, and LLC-MK2 cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher PO2, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a PO2 greater than 40 mm Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a PO2 insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher PO2 for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that of a comparable population of fibroblasts for which low oxygen may enhance survival and proliferation.

Light-induced chromatid damage in human skin fibroblasts in culture in relation to their neoplastic potential.

Skin fibroblasts from ataxia telangiectasia and xeroderma pigmentosum (XP) donors and from the XP sib (possible heterozygote), all genetically predisposed to a high risk of cancer, show an increased susceptibility to light-induced chromatid breaks after culture in vitro. Light-induced chromatid breaks were shown previously to result from generation of hydrogen peroxide (H2O2) during light exposure. The level of susceptibility attained is significantly higher than that observed in 13 lines of fibroblasts from normal skin of donors ranging in age from 3 days to 92 years or from fetal skin tested at various population doubling levels. Two lines of normal skin fibroblasts transformed by chemical carcinogens to neoplastic cells also show a significant increase in susceptibility as compared with their untransformed controls. These data indicate for human cells, as reported earlier for mouse cells, an association between enhanced susceptibility to light-induced chromatid damage and neoplastic potential; this association is further supported by the high susceptibility of cells derived from a human adenocarcinoma. Two observations are consistent with the concept that the increased susceptibility does not result from greater initial damage to the DNA of the neoplastic cells. First, activities of the ubiquitous H2O2 scavenging enzyme, glutathione peroxidase, are similar in the paired normal and neoplastic cell populations. Second, cells of the paired lines are equally sensitive to DNA breakage by exogenous H2O2. The enhanced susceptibility associated with neoplastic potential may result from an impaired capacity to repair DNA rather than a greater initial sensitivity of the neoplastic cells to the damaging agent.

Effect of visible light on benzo(a)pyrene binding to DNA of cultured human skin epithelial cells.

The ubiquity of the photosensitive carcinogen benzo(a)pyrene (BP) and visible light in the environment suggests that their interaction might lead to photoproducts harmful to humans. To test the combined impact of these two agents on human epithelial cells, binding of BP to cellular DNA was assessed following treatment of cultures with BP and low-intensity (4.6 watts/sq m) intermittent (12 hr daily, 3 to 5 days) cool white fluorescent light. Light exposure reduced the formation of covalent BP adducts 20-fold (from 150 to 7 pmol BP per mg DNA) in cells treated with 1 microgram BP per ml and completely inhibited cytotoxicity; even with 10 microgram BP per ml, light exposure markedly inhibited cytotoxicity. However, at low BP dosage (0.1 microgram/ml), covalent adducts (2 pmol/mg DNA) to cellular DNA are produced and their formation is not influenced by light. These adducts persisted for at least 7 days following treatment; this observation suggests that chronic low-level exposure of human epithelium to BP may lead to an accumulation of DNA damage.

Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts. Role of hydrogen peroxide and related free radicals.

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.

A new culture medium for human skin epithelial cells.

A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1:1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 microgram per ml and 5 microgram per ml, respectively, did not appreciably enhance the growth of the epithelial cells.

Population density as a factor in the evolution of neoplastic cell lines.

The influence of population density in the progression from the nonneoplastic to the neoplastic state has been reassessed. Two twice-cloned, nonneoplastic mouse lines, NCTC 7914 and 7915, were transferred each 3 to 4 days at inoculum sizes selected to minimize or maximize cell-cell contact, 1 X 10(5) or 4 X 10(5) cells/T-15, respectively. As tested by in vivo assay, the regime designed to minimize cell-cell contact did not reproducibly delay transformation, and tumor production was observed in all lines, irrespective of inoculum size. Also, results of tumorigenesis assays correlated with blind evaluation of morphological and cytological alterations, growth in agarose, and susceptibility to killing by activated macrophages. Generally higher saturation densities were seen as a function of period in culture, and no significant differences in glucose utilization or lactic acid production were observed between nonneoplastic and neoplastic cell populations.

Density-dependent effects of oxygen on the growth of mammalian fibroblasts in culture.

Various concentrations of oxygen were used to determine the optimum culture medium PO2 for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T-15 flasks were seeded with less than or equal to 2 X 10(4) cells (less than or equal to 1.3 X 10(3) cells/cm2), the highest plating efficiencies and cell yields were obtained with a culture medium PO2 of 40-60 mm Hg. At higher inoculum sizes (10(5) cells per T-15) used routinely for mass cultured, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% O2 (growth medium PO2 approximately equal to 125-135 mm Hg) and those gassed with 1% O2 (growth medium PO2 approximately euqal to 40-60 mm Hg). The enhanced clonal growth observed at the latter PO2 results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% O2. A slight extension of lifespan was observed in WI-38 cells serially subcultured with a gas phase of 1% O2.