E1A Oncogene-induced Sensitization of Human Tumor Cells to Innate Immune Defenses and Chemotherapy-induced Apoptosis in Vitro and in Vivo

Cell Lines.

MDA-MB435S is a human ductal carcinoma line derived from a patient with metastatic disease who had received no previous chemotherapy (37) . These cells have a p53 gene rearrangement that causes reduced expression of p53 (38) and both primary tumors and lung metastases in nude mice (2 , 39) . Saos-2 is a human osteosarcoma cell line that has a genomic deletion that eliminates the p53 gene (40) . Saos-2 is inherently resistant to NK killing and converted to NK susceptibility by E1A expression (16) . E1A-positive Saos-2 were used only as an NK susceptible cell control in these experiments. These cell lines were obtained from the American Type Culture Collection. H4 is a subclone of the human HT1080 fibrosarcoma cell line that expresses only mutant p53 (41 , 42) . The E1A oncogene encodes two families of products, one derived from a 13S mRNA and one from a 12S mRNA (hereafter referred to as E1A 13S or E1A 12S). E1A expression in H4 has been shown to convey susceptibility to killing by both NK cells and TRAIL (12 , 14) . MB435S and Saos-2 cell lines that express E1A 12S and an H4 cell line that expresses genomic E1A were provided by S. Frisch (20 , 41) . MB435S clones stably expressing genomic E1A were established after transfection with the plasmid p1A-pac and selection in puromycin (16) . All cell lines were maintained by weekly passage in DMEM containing antibiotics and 5% calf serum (Sterile Systems, Logan, UT). Medium for E1A-expressing, G418-resistant cells was supplemented with 200 μg/ml this antibiotic (Life Technologies, Inc., Grand Island, NY). Medium for puromycin-resistant cells was supplemented with this inhibitor (Sigma Chemicals, St. Louis, MO) at a final concentration of 5 μm. All cell lines were tested for contamination with Mycoplasma using the Mycotect assay (Life Technologies, Inc.) and were negative. Levels of E1A oncoprotein expression in different cell lines were compared by analyzing equal amounts of cellular proteins by Western analysis using the E1A-specific monoclonal antibody M73 (43) , as described (14) .

Apoptosis and Cytotoxicity Assays.

Assays of NK cell-induced killing of tumor cells were done as described (44) using ficoll-diatrizoate-separated human blood mononuclear cells from normal donors or the human NK cell line YT (45) , which was provided by G. Cohen. Bone marrow-derived macrophages were obtained from the femurs and tibias of mice and cultured in DMEM supplemented with 15% granulocyte macrophage colony-stimulating factor prepared from transfected Chinese hamster ovary cells provided by T. Potter. Granulocyte macrophage colony-stimulating factor was removed from the culture medium 48 h before cytolysis assays. Twenty-four h before cytolysis assays, macrophages were activated with IFN-γ (100 units/ml; R&D Systems, Minneapolis, MN) and lipopolysaccharide (1 μg/ml; Sigma). Target cells were labeled with ³H-thymidine, and 48-h cytolysis assays were performed as described (5) . The mean percentage spontaneous radiolabel release from all types of target cells was <20%.

Assays of Fas-induced apoptosis were done using antihuman Fas antibody (BD-PharMingen, San Diego, CA) immobilized on plastic surfaces as described (46) . Assays of TRAIL-induced apoptosis were done as described (14) using recombinant human TRAIL (R&D Systems). The chemotherapeutic drugs etoposide, cisplatin, cytarabine, and hydroxyurea (Sigma) were prepared as stock solutions in DMSO and diluted into culture medium just before each apoptosis assay. Initial dose-response assays defined optimal, apoptosis-inducing drug concentrations and excluded residual DMSO in assay medium as a factor in cytotoxicity.

The apoptotic nature of the cell death response of E1A-positive cells to both chemotherapeutic and immunological injuries has been defined (47) . The number of cells exhibiting characteristic nuclear chromatin condensation was quantitated morphologically by counting cells stained with ethidium bromide (48) . The amount of radiolabeled, low molecular weight DNA released from injured cells was used as a second measure of apoptosis-related DNA fragmentation using the method of Duke and Cohen (48) . All apoptosis assays were done in DMEM containing antibiotics and 10% endotoxin-free fetal bovine serum (Sterile Systems).

Tumor Induction Assays.

Cohorts of 8-week-old female nude mice (athymic NCr-nu) were obtained from the Frederick Cancer Research Facility or Taconic Laboratories and maintained in isolator cages. Quantitative tumor induction studies were done as described (49) , by inoculating animals s.c. between the scapulae with serial 10-fold concentrations of cells ranging from 10⁴ to 10⁸ cells/inoculum (depending on the experiment) in a 0.2-ml volume. Cells were prepared by trypsinization of subconfluent cell monolayers followed by prompt trypsin inactivation with serum, repeated washing, and resuspension in additive-free, serum-free DMEM for injection.

Treatment of nude mice with etoposide (Sigma) was done using an optimal dose defined previously and treatment schedule, based on LD₅₀ data and experience with experimental therapy of other tumor lines (50 , 51) . Etoposide was prepared in a stock solution in DMSO and then diluted into HBSS at a final drug concentration of 750 μg/ml just before treatment of each mouse group. Each animal was weighed and treated with a dose calculated to deliver 30 mg/kg etoposide per i.p. injection. Treatment was started 1 day after tumor challenge and repeated approximately every 4 days throughout the period of observation. Sham-treated animals were treated with the same volumes and dosing schedules using HBSS containing an equal concentration of DMSO to that used for etoposide treatments.

Animals were checked twice weekly for tumor development and sacrificed when tumors measured >20 mm in mean diameter or at the end of the period of observation if tumor free. The 50% end points of tumor production (TPD₅₀s) were estimated by the method of Karber (52) at each time point. TPD₅₀s were converted into survival data to reflect the percentage of tumor-free animals as described (36) using Sigma Plot software (SPPS). Average tumor latency was calculated across all cell challenge doses using JMP software (SAS Institute) for nonlinear curve fitting and estimation of the time required to reach 50% tumor development for each cohort of mice.

Tumors were randomly selected in each experiment for testing of E1A protein expression. Tumors were minced and trypsinized, and cells were tested by immunoblotting with E1A-specific monoclonal antibody at the first tissue culture passage.

The significance of the differences in the survival curves for mice in different cell challenge and treatment groups was estimated using the Kaplan-Meier method and Wilcoxon test with JMP software.

E1A Expression Sensitizes p53 Mutant Human Breast Cancer Cells to Immune-mediated Apoptosis.

MB435S human breast cancer cells were chosen for these studies for several reasons. These cells have a p53 mutation that results in low expression of a mutant tumor suppressor product that is functionally defective (53) . MB435S is therefore typical of ∼50% of breast cancers with p53 mutations or deletions (38) . MB435S also expresses low levels of both HER-2/c-erbB-2/neu-encoded p185 and mdr-1-encoded p170, both of which can cause tumor cells to be resistant to chemotherapeutic drugs (54) . To our knowledge, the ability of p185 and p170 to influence immunologically mediated injury has not been evaluated. Use of MB435S simplified the interpretation of E1A effects on cytolytic susceptibility, because it was possible to eliminate overexpression of both p185 and p170 proteins as factors in cellular resistance to chemotherapeutic and immunologically mediated injuries. Initial studies also showed that MB435S cells are inherently resistant to apoptosis triggered by human and rodent NK cells and a variety of chemotherapeutic drugs, 5 making MB435S suitable for studies of E1A-induced conversion from the cytolytic resistant to the cytolytic susceptible phenotype.

MB435S cells expressing either genomic E1A (i.e., both E1A 12S- and 13S-encoded proteins; MB435S-E1A.1, MB435-E1A.2) or only E1A 12S products (MB435-E1A12S.1) were tested for E1A oncoprotein expression by immunoblotting with an E1A-specific monoclonal antibody (Fig. 1, A and B) ⇓ .

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Fig. 1.

E1A oncoprotein expression as detected by immunoblotting. A, E1A expression by MB435 cell lines. B, comparative expression of E1A 12S protein by a parental MB435 clone expressing E1A 12S cDNA that was used for nude mouse tumor challenge versus two different tumor cell lines tested at the first tissue culture passage from two different, progressive nude mouse tumors arising on etoposide-treated mice (nm1 and nm2) challenged with 10⁶ MB435-E1A12S.1 cells. C, E1A expression by Saos-2 cells stably transfected with 12S cDNA. In each case, an equal amount of protein from E1A-negative, parental cells was loaded in the first lane.

E1A-positive MB435S cell lines were tested for sensitivity to lysis by NK cells and activated macrophages and to apoptosis triggered by cell surface Fas or TRAIL death receptors (Figs. 2 ⇓ and 3) ⇓ . The E1A-induced increase in susceptibility of p53 mutant MB435S cells to killing by NK cells was comparable with that observed with p53-negative Saos-2 (Fig. 2A ⇓ ; Refs. 16 and 40 ). E1A-induced cytolytic susceptibility of MB435S was also detected using the continuous human NK cell line YT (Fig. 2B ⇓ , left panel) and cytokine-activated, bone marrow-derived mouse macrophages (Fig. 2B, right panel). These results are consistent with our observation that there is no species restriction of NK or macrophage killing of E1A-positive cells (5 , 34) .

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Fig. 2.

E1A-induced cytolytic susceptibility to killer cells. A, comparison of susceptibility of E1A 12S-expressing MB435 and Saos-2 cells to killing by human blood NK cells. E1A-positive cells of both types were significantly more sensitive than E1A-negative cells to NK killing across the range of lymphocyte to target cell ratios (P < 0.05). B, comparison of susceptibility of E1A-negative versus E1A 12S-positive MB435 cells to killing by the human NK cell line YT, at a 50:1 NK:target cell ratio (left panel) or by monolayers of bone marrow-derived mouse macrophages activated with IFN-γ and lipopolysaccharide (right panel). Results represent the mean ± SE of three different assays. E1A-positive cells were significantly more susceptible to both types of killer cells (P < 0.05).

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Fig. 3.

E1A-induced cytolytic susceptibility to Fas- and TRAIL-dependent killing. Results represent the mean ± SE of two (anti-Fas antibody; A) or three (TRAIL; B) apoptosis assays. E1A-positive cells were significantly more sensitive to apoptosis triggered by both agents (P < 0.05).

When cell surface Fas receptors were cross-linked with anti-Fas antibody, E1A-negative MB435S cells exhibited no morphological changes or evidence of cell death. In contrast, anti-Fas antibody treatment induced apoptosis in E1A-positive MB435S cells (Fig. 3A) ⇓ . Similar E1A-induced apoptosis sensitivity was observed when cells were treated with recombinant TRAIL (Fig. 3B) ⇓ . Cell death after Fas- and TRAIL-induced signaling was confirmed to be apoptotic by two independent assays: (a) morphological assessment of nuclear chromatin condensation and (b) quantitation of DNA fragmentation (data not shown).

E1A Induces Chemosensitivity Independently of Tumor Cell Expression of p53.

The chemosensitivity of MB435S cells expressing either genomic E1A (i.e., both E1A 12S- and 13S-encoded proteins) or only E1A 12S products (Fig. 1A) ⇓ was characterized using the proapoptotic topoisomerase II inhibitor etoposide (Fig. 4A) ⇓ . In studies of E1A-expressing mouse fibroblasts, chemotherapy drug-induced apoptosis has been reported to depend on expression of p53 (16 , 55) . In contrast, p53 expression is not required for E1A-induced cytolytic susceptibility of different types of human tumor cells (15 , 21 , 23 , 47) . In our experiments, E1A-positive MB435S, which express reduced levels of p53 because of gene rearrangement, were significantly more sensitive to the cytotoxic effect of etoposide than were E1A-negative cells (Fig. 4A) ⇓ . E1A 12S expression alone (MB435-E1A.12S) was as effective at sensitizing cells to etoposide as expression of both E1A 12S and 13S transcripts (MB435-E1A.l and MB435-E1A.2). The apoptotic nature of the cell death response by E1A-positive cells was confirmed by counting the numbers of cells exhibiting nuclear chromatin condensation and fragmentation and also by quantitating the release of low molecular weight DNA from the nuclei of injured cells (Fig. 4B) ⇓ . To further assess the range of chemotherapeutic drug injuries to which E1A-positive, p53-negative human tumor cells are susceptible, etoposide-induced apoptosis of p53-negative Saos-2 cells was compared with E1A-related sensitivity to apoptosis triggered by three other chemotherapeutic agents from different drug classes (cisplatin, an alkylating agent, and cytarabine and hydroxyurea antimetabolites; Fig. 4C ⇓ ). All three classes of drugs induced apoptotic cell death of p53-negative, E1A-positive cells that was significantly greater than that observed with E1A-negative cells (P < 0.05).

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Fig. 4.

E1A-induced chemosensitivity of MB435 and Saos-2 cells. A, comparison of the sensitivity of MB435 cells expressing either genomic E1A (-E1A.1 and -E1A.2) or E1A 12S cDNA versus E1A-negative control cells to etoposide-induced apoptosis. Results represent the mean ± SE of three different assays. All three E1A-positive cell types were significantly more sensitive to etoposide across the range of drug concentrations tested (P < 0.05). B, comparison of three different indicators of etoposide-induced cell death of E1A-negative (white bars) or E1A-positive (black bars) MB435. Results represent the mean ± SE of three different assays of each type. E1A-positive cells were significantly more sensitive to etoposide-induced apoptosis (P < 0.05). C, comparison of the sensitivity of E1A 12S-expressing, p53-negative Saos-2 cells versus E1A-negative control cells to apoptosis induced by four different chemotherapeutic drugs. Results represent the mean ± SE of four different apoptosis assays using the optimal concentration of each drug, as determined in dose-response studies. E1A-positive cells were significantly more sensitive to apoptosis induced by all drugs (P < 0.05).

E1A Expression Sensitizes Human Tumor Cells to Innate Immune Defenses and Chemotherapy in Nude Mice.

Primary objectives of these studies were to compare the tumorigenicities of E1A-positive and -negative human tumor cells in the context of the innate immune defenses and to contrast the tumorigenicity of E1A-positive cells in untreated mice versus mice treated with an apoptosis-inducing chemotherapeutic agent. We first considered whether there were any detectable in vitro differences in MB435 cells caused by E1A expression that might confound the interpretation of tumor induction experiments. Studies of in vitro growth characteristics showed subtle changes in cell morphology but no significant differences in cell doubling times of E1A 12S-positive versus E1A-negative MB435S cells, consistent with reported observations (20 , 25) . Therefore, the E1A-positive cells did not have either an inherent in vitro growth disadvantage or advantage compared with E1A-negative cells. We next asked whether E1A expression persisted in MB435 cells after passage through nude mice. It had been reported that nude mouse tumor formation by E1A-positive human melanoma cells resulted in selection for cells that had lost E1A protein expression detectable by immunoblotting, despite persistence of E1A gene sequences in the cells (56) . In our studies, cells grown in vitro from excised tumors developing on nude mice challenged with E1A-positive MB435 cells expressed levels of E1A protein comparable with the E1A levels detected in the cells used for the original inoculation (e.g., see Fig. 1B ⇓ ; MB435-E1A12S.1 = challenge cell versus MB435-E1A12S.1-nm1 and -nm2 = tumor cells from nude mice challenged with MB435-E1A12S.1). Therefore, tumor formation by E1A-expressing cells was not explained by selection for E1A-negative variants within the E1A-positive MB435 cell population inoculated into mice.

The effect of E1A expression on tumor development was tested by observing nude mice for 3 months after inoculation with either E1A-positive or -negative MB435S cells. In some experiments, mice challenged with both cell types were treated with chemotherapy and compared with sham-treated controls. Previous studies using these cells (20) or other E1A-expressing human tumor cells (57) showed that nude mouse tumors induced by E1A-positive cells developed more slowly than tumors induced by E1A-negative controls during the 1^st month of observation but did not reveal the long-term outcome of comparative tumor development.

To test the effects of chemotherapy on tumor development, cohorts of mice were divided into four groups of 6, and each group was inoculated with 10⁴, 10⁵, 10⁶, or 10⁷ cells/mouse using MB435S or MB435-E1A12S.1 cells and then split into two groups of 3; one group of 3 was sham treated by i.p. inoculation of HBSS, and the other group was treated with HBSS containing etoposide (30 mg/kg/mouse) as described in “Materials and Methods.” Three parameters of tumor development were measured: (a) time to first detection of s.c. tumors; (b) time to 50% tumor incidence calculated across the whole cohort of challenged mice; and (c) threshold cell dose required for tumor production (final TPD₅₀; Refs. 36 ). Each experiment was repeated in a different mouse cohort using the identical protocol.

Nude mice were significantly more resistant to challenge with E1A-positive cells than with E1A-negative cells by Kaplan-Meier analysis (Fig. 5 ⇓ ; solid circles versus solid triangles, respectively, and table, column 3 versus column 1, respectively; P < 0.05). The lag time between s.c. inoculation and the first tumor detected in animals challenged with 10⁷ cells increased from 6 days for E1A-negative cells to 35 days for E1A 12S-expressing cells in sham-treated mice (Fig. 5 ⇓ , table). The average tumor latency calculated across all cell challenge doses increased from 11 days for E1A-negative cells to 41 days for E1A-positive cells (Fig. 5 ⇓ , table). This estimation of the average latency period provides a more reproducible measure of tumor latency than the time of first tumor appearance in a single animal, because it averages variations across the entire animal cohort challenged with a range of cell doses (35) . E1A expression also slightly reduced the efficiency of tumor induction (threshold cell dose), as evidenced by a doubling in the number of challenge cells required to reach the final 50% end point of tumor production (TPD₅₀ = 4.8 versus 4.5, respectively). This trend toward reduced tumor-inducing efficiency of E1A-positive MB435S cells was confirmed in a second experiment in another mouse cohort where there was a 5-fold increase in the number of challenge cells needed to reach the TPD₅₀ end point (E1A-positive cells = 5.2, E1A-negative cells = 4.5; 0.7 log = 5-fold difference). Our experience in many nude mouse tumor induction experiments is that differences in TPD₅₀ of <0.5 logs are usually not significant. This is consistent with the findings in these experiments in which the mean SD ± TPD₅₀ from four different challenges of nude mice with E1A-positive MB435S cells was 5.2 ± 0.5 cells/mouse. In summary, these data showed that the cumulative innate immune defenses of nude mice delayed tumor formation by breast cancer cells expressing E1A protein as evidenced by Kaplan-Meier analysis and prolongation of the average tumor latency periods but had minimal effects on the long-term outcome of tumor development as evidenced by minor effects on net tumor-inducing efficiency (TPD₅₀) relative to E1A-negative parental, breast cancer cells.

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Fig. 5.

Comparative MB435 tumor development in nude mice expressed as survival curves after s.c. challenge with E1A-negative (inverted triangles) or E1A-positive (circles) cells. Mice were either sham treated (closed symbols) or treated (open symbols) with twice weekly i.p. injections of etoposide (30 mg/kg). Points represent the mean percentages of tumor-free mice estimated by calculations across the entire cell dose range. Each curve represents the kinetics of tumor development in a cohort of 12 mice. Comparative tumor parameters are listed in the table below.

Etoposide treatment of tumor-challenged nude mice was used to test the in vivo correlation of the E1A-induced chemosensitivity of breast cancer cells (Fig. 5 ⇓ , open symbols and table, columns 2 and 4). Etoposide treatment significantly delayed tumor development by E1A-expressing cells (open circles) when compared with tumor development in all nude mice challenged with E1A-negative cells by Kaplan-Meier analysis (P = 0.02). This E1A-induced sensitivity to chemotherapy also was reflected in an increased time to first tumor appearance (50 versus 35 days) and average tumor latency calculated across the cohort (59 versus 41 days; Fig. 5 ⇓ , table). In contrast, etoposide treatment had no significant effect on tumor development by E1A-negative cells (Fig. 5 ⇓ open versus closed triangles and table, columns 1 and 2). Chemotherapy further reduced the tumor-inducing efficiency of E1A-positive cells by 2.5-fold, compared with sham-treated mice (increased TPD₅₀ from 4.8 to 5.2), but had no effect on the tumor-inducing efficiency of E1A-negative cells (TPD₅₀ = 4.5 in both cases). A similar, E1A-related enhancement of the etoposide effect on the tumorigenicity of MB435S cells was observed in a second experiment (increased average tumor latency from 29 to 51 days and TPD₅₀ from 5.1 to 5.5).

Similar studies of sham- and etoposide-treated nude mice were repeated using H4 fibrosarcoma cells. E1A expression does not change the in vitro growth rate of H4 (41) but does convert them from a cytolytic resistant to a cytolytic susceptible phenotype when challenged by NK cells or TRAIL (12 , 14) . E1A effects on the chemosensitivity of H4 cells followed the predicted pattern of E1A-induced susceptibility. The mean ± SE percentage of killing in seven cytotoxicity assays with etoposide at 25 μm was: E1A-negative H4 = 12.9 ± 3.3% versus E1A-positive H4 = 59.1 ± 4.1% (P < 0.001).

Quantitative tumor induction studies showed that E1A-induced changes in H4 tumorigenicity were similar to those observed with MB435S cells (Fig. 6) ⇓ . E1A expression significantly delayed H4 tumor development in sham-treated nude mice compared with E1A-negative H4 (Fig. 6 ⇓ , solid circles versus solid triangles, respectively; P = 0.03 by Kaplan-Meier analysis) and was associated with a doubling of the average tumor latency of the challenged cohort (15 versus 7 days, respectively). E1A-positive cells also had a 10-fold lower tumor inducing efficiency than E1A-negative H4 cells (TPD₅₀ = 5.2 versus 4.2, respectively; Fig. 6 ⇓ , table, column 3 versus column 1, respectively). Etoposide treatment significantly delayed tumor development in animals challenged with E1A-positive H4 compared with mice challenged with E1A-negative cells (Fig. 6 ⇓ , open circles versus open triangles, P = 0.03 by Kaplan-Meier analysis) and further prolonged the average tumor latency of the treated cohort (29 days versus 15 days, respectively) and reduced the tumor-inducing efficiency of E1A-positive H4 cells (TPD₅₀ = 5.8) compared with E1A-negative H4 cells (TPD₅₀ = 4.5) by 20-fold (Fig. 6 ⇓ , table, column 4 versus column 2, respectively).

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Fig. 6.

Comparative H4 tumor development in nude mice expressed as survival curves after s.c. challenge with E1A-negative (inverted triangles) or E1A-positive (circles) cells. Mice were either sham treated (closed symbols) or treated (open symbols) with twice weekly i.p. injections of etoposide (30 mg/kg). Points represent the mean percentages of tumor-free mice estimated by calculations across the entire cell dose range. Each curve represents the kinetics of tumor development in a cohort of 12 mice. Comparative tumor parameters are listed in the table below.

E1A Does Not Eliminate the Tumorigenicity of Human Cells in Animals with Innate Immune Defenses.

In both the human breast cancer and fibrosarcoma cell systems tested here, high-level E1A expression caused a significant delay in tumor formation and, to a variable extent, a reduction in tumor-inducing efficiency (Figs. 5 ⇓ and 6) ⇓ . E1A expression did not, however, eliminate tumor induction by either human tumor cell line, if the cells were inoculated at a sufficient dose and the animals were observed for prolonged periods of time after challenge.

The question of whether E1A-expressing human tumor cells can induce tumors in nude mice has been a source of debate. It has been suggested that E1A expression in primary human cells may induce limited cell cycle progression but may be unable to transform cells to a fully neoplastic and tumorigenic phenotype (58) . The related postulate is that E1A induces human cells to exhibit a relatively normal epithelial-like phenotype, thus eliminating their tumorigenic potential (59) . There are, however, a few reports that E1A-expressing, human tumor cells or immortalized human cell lines are tumorigenic in immunodeficient mice (60, 61, 62) .

The studies presented in this report suggested a possible explanation for these discrepancies. The results indicated that E1A expression might both prolong the time for tumor development and reduce the tumor-inducing efficiency of a human tumor cell in a host like a nude mouse with an intact innate immune response. These observations predicted that E1A-immortalized human cells might form tumors very inefficiently in nude mice, in part because of E1A-induced cytolytic susceptibility and the innate immune defenses of the animals. The results of our previous studies also predicted that the level of NK cell response of the challenged animal might determine the success of the tumor challenge with E1A-positive human cells (32 , 33 , 63) , e.g., nude rats that have higher levels of NK activity than nude mice (33) might be more resistant to challenge with NK-susceptible, E1A-expressing human cells.

To test these possibilities, nude mice and rats were challenged s.c. with large numbers (10⁶, 10⁷, or 10⁸) of E1A-expressing human 293 cells and observed for 3 months for tumor development (Fig. 7) ⇓ . 293 is an immortalized human cell line that was derived by transfection of normal human embryonic kidney cells with sheared adenoviral DNA but that may be of neural cell origin (64) . 293 express high levels of E1A oncoprotein and are highly NK sensitive (5) . Small tumors were first apparent 3–4 weeks after 293 cell inoculation into both types of animals. Nude mice developed tumors only after inoculation of 10⁷ or 10⁸ cells, with a calculated TPD₅₀ of 6.5 cells. Nude rats were slightly more resistant to tumor formation, with a TPD₅₀ of 7.2 cells. In both types of animals, the last tumors were detected 6 weeks after cell inoculation. Therefore, a challenge with 3–15 × 10⁶ 293 cells and an observation period of ≥4–6 weeks was required to detect tumor formation in these experiments. These data suggested that, like E1A expression in human breast cancer and fibrosarcoma cells, E1A expression during immortalization of primary human cells may limit the tumorigenicity of these cells in NK competent animals but that this limitation can be overcome with high cell challenge numbers. The significance of the apparent relative resistance of nude rats versus nude mice to 293 challenges could not be determined in this limited study.

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Fig. 7.

Comparative 293 tumor development in nude mice and nude rats expressed as survival curves after s.c. challenge. Points represent the mean percentages of tumor-free mice estimated by calculations across the entire cell dose range. The 50% end points for tumor induction (TPD₅₀) at the end of the 3-month observation period were 6.5 for nude mice and 7.2 for nude rats. The curves represent the kinetics of tumor development in cohorts of 16 nude mice (4 mice each challenged s.c. with 10⁸, 10⁷, or 10⁶ cells) and 12 nude rats (3 rats each challenged s.c. with 10⁸, 10⁷, or 10⁶ cells).