MOLECULAR CARCINOGENESIS 6:252-259 (1992)

Partial Suppression of Tumorigenicity in a Human Lung Cancer Cell Line Transfected With Krewl Jorge Caamano. Marion DiRado, Toshihiko lizasa, Shigeru Momiki, Elma Fernandes, Curt Ashendel, Makoto Noda, and Andres J. P. Klein-Szanto'

Department of Pathology (JC, MD, TI,SM,AJPK-S), Fox Chase Cancer Center, Philadelphia, Pennsylvania; Department of Medicinal Chemistry (EECA), Purdue University, West Lafayette, Indiana; and Department o f Viral Oncology (MN), Cancer Institute, Tokyo, Japan A human non-small-cell lung carcinoma cell line, Calu-6 (from an anaplastic carcinoma), was transfected with t h e Ki-ras-related anti-oncogene Krev-1. Several transfectant lines were obtained t h a t showed a reduced tumorigenicity in nude mice w i t h respect to t h e parental and control transfected cell lines. This decrease was approximately 50% in t u m o r incidence a t 4 wk after subcutaneous inoculation of t h e transfected cells. In addition, t h e volume of t h e Calu-6 revertant-derived tumors was three t o 10 times smaller t h a n t h a t of t h e equivalent tumors produced by inoculation of t h e control cell line transfected w i t h t h e neomycin-resistance gene. Krev-1-transfected cells t h a t exhibited reduced tumorigenicity expressed Krev-1 mRNA and had variable numbers o f copies of t h e Krev-1 gene. Moreover, Krev-1-transfected cells exhibited a more differentiated squamous epithelial morphology than t h e parental and control cell lines did. Moderately elevated levels of protein kinase C activity were detected in some revertant clones. Such activity correlated w i t h t h e level of expression of Krev-1 mRNA in most cases. In summary, Krev-1 induced important morphological and biological changes in transfected Calu-6 cells t h a t w e interpreted as partial reversion of the malignant phenotype. o 1992~ i ~ e y - ~ iInc. ss, Key words: Revertants, t u m o r suppression, human lung carcinoma INTRODUCTION Reversion of the neoplastic phenotype in human cancer cells has been demonstrated by fusion of normal and neoplastic cells [II, by chromosome transfer [2,31, and by transfection with different tumor suppressor genes [4,51. Recently, several groups have been successful in restoring the expression of the retinoblastoma-susceptibility (Rbf [4,6-91 and p53genes [5,10,1 I]in cell lines derived from tissues of diverse origin. The Krev-I gene, also known as Rap 1A or Smg-p21A, is a putative tumor suppressor gene isolated from a human cDNA expression library by its ability to produce morphologically untransformed revertants when transfected into Kirsten sarcoma virus-transformed NIH 3T3 cells [I2-1 41. The Krev-I gene product belongs to the group of guaninenucleotide binding proteins and shares 53% amino acid identity with the ras proteins. Studies using mutant Krev-I genes as well as chimeric Krev-1/Ha-ras genes have revealed that a region homologous to the putative effector-binding domain of ras proteins and an adjacent region N-terminal to this domain are responsible for the tumor-suppressor characteristics of this gene [ 15,161. Moreover, it has been found that Krev-I protein in the GTP form is able to bind to ras GTPase-activating protein (GAP) [ 171, thus interfering with ras GAP binding to ras protein. Despite the known interaction between p2 1 Krev and ras GAe the exact mechanism of action of Krev-I with respect to the ras pathway is not entirely clear. Nevertheless, recent evidence in Drosophila supports the opposing and competitive actions of these two proteins [ 18,191. Recently, a Krev-specific GAP 0 1992 WILEY-LISS, INC,

was cloned [20,21]. It binds to p21 Krev-I protein in the GTP or GDP form. Under growth conditions, Krev GAP would maintain p21 Krev in the GDP-bound form. Activated rasgenes, especially Ki-ras, are found in approximately30-50% of human non-small-cell lung carcinomas. Patients with these tumors have a poor prognosis and shorter survival time than patients with tumors containing normal rasgenes [22-241. This high incidence of mutations in the ras genes, especially in Ki-ras, indicates that ras has an important role in tumor development. Since to our knowledge no reports have been published on the effect of Krev-I transfection in human carcinoma cells, we attempted to investigate whether Krev-I would be able to revert the malignant phenotype of human lung tumor cells carrying mutations in the Ki-rasgene. For this purpose, a human non-small-cell lung carcinoma cell line, Calu-6, was transfected. This cell line was derived from an anaplastic carcinoma of the lung and carries a Ki-rasgene activated by a point mutation in codon 61 (GlnLys) [25]. Several G418-resistant clones were isolated and assayed for Krev-I mRNA expression as well as in vitro growth and tumorigenicity.

'Corresponding author: Fox Chase Cancer Center, 7701 Burholrne Ave., Philadelphia, PA 191 1 1 . Abbreviations: GAP. GTPase-activating protein; PKC, protein kinase C; Rb, retinoblastoma-susceptibilitygene; SSC. standard saline citrate; TPA, 12-0-tetradecanoylphorbol- 1 3-acetate.

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MATERIALS AND METHODS Cells Calu-6 cells, derived from a human anaplastic carcinoma, were obtained from the American Type Culture Collection (Rockville, MD) and maintained in antibiotic-free minimal essential medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum in an atmosphere containing 5% COz. Transfections pKrev-I, which contains the Krev-I cDNA sequence under the control of the SV40 early promoter and also harbors the neomycin-phosphotransferase gene [ I 41, was transfected by lipofection using the Lipofectin kit (GIBCOBRL, Gaithersburg, MD) into Calu-6 cells. Control transfections were performed in parallel using the same cell lines and the pSV2 construct, which carries only the neomycin-phosphotransferasegene. Forty-eighthours after transfection, the cells were treated with 0.7 mg/mL G418 (Sigma Chemical Co.) and incubated for 1-3 w k at 37°C with the medium changed every second day. After the colonies reached 5 mm in diameter, they were harvested and expanded in larger flasks. Southern and Northern Analysis DNA was isolated from the cultured cells using the proteinase K-phenol extraction method [261. DNA digested with BamHl was separated by electrophoresis in 1% agarose gels. Total RNA was extracted from each cell line by an acid guanidium isothiocyanate-phenol chloroform extraction method [261 and electrophoresedin 1YOagarose2.2 M formaldehyde gels [26]. Both RNA and DNA were transferred to nylon filters and hybridized to random-primer 32P-labeledprobes [27]. A 1.25-kb Rsal fragment containing the coding region and the 3’ end of the human Krev-I gene was used as a probe for hybridization [14]. Dot Blot and Quantitative Radioanalysis Three micrograms of genomic DNA was resuspended in 50 pL. of TE, pH 8.0; 5 p.L of 3M NaOH was then added to each sample, and they were incubated at 65°C for 1 h. Once the samples reached room temperature, they were diluted to a volume of 200 pL with 6 x standard saline citrate (SSC; 6 x SSC = 0.9 M NaCl and 0.09 M sodium citrate, pH 7.0). The samples then were spotted onto a nylon membrane (Nytran; Schleicher & Schuell, Inc., Keene, NH) using a Hybri-Slot 24-well filtration manifold (GIBCO-BRL). The blot was baked for 2 h at 80°C and subsequently hybridized to the Krev-I cDNA probe and to a human p-actin cDNA probe for normalization of DNA loading. An Ambis Radioanalytic Imaging System (Scanner Mark 11, software version 1.83; San Diego, CA) based on gas ionization using a 90% argon-10% methane mixture as detector gas/ quenching agent was used to measure the P-particle emission counts. The results were quantitated above an experimental and instrument background averaging 0.026 cpm/mm2.Detection time was 420 min.

Protein Kinase C Activity The protein kinase C (PKC) activity in the soluble extracts of the Calu-6 parental and revertant cell lines was measured as described earlier [281 with the following modifications. Fifteen to fifty micrograms of protein made soluble with 3-([3-cholamidopropyl]dimethylammonio)-l-propanesulfonate was assayed for 10 min at 30°C in 20 mM p-nitrophenyl phosphate, 40 m M [y3*P]ATt and 10 m M MgClz in 20 m M Tris, pH 7.4, using 480 p g h L type 111 histone (United States Biochemical Corp., Cleveland, OH) as substrate. The PKC activity (in pmol/min/mg protein) was determined as the difference between the amount of radiolabeled phosphate transferred to the substrate in the presence of 80 pg/mL phosphatidylserine, 600 ngimL 12-0-tetradecanoylphorbol-I3-acetate (TPA), and 0.5 m M CaClz and in the absence of phospholipid, TPA, and calcium and the presence of 1 m M EGTA. The PKC activity of each independent sample of each cell type in a single experiment was then normalized with respect to the average of duplicate controls. Labeling Index Incorporation of radiolabeled DNA precursors into control and transfected Calu-6 cells was accomplished by incubating the cells for 45 min in culture medium containing 20 pCi/mL tritiated thymidine. After fixation in formalin, the cells were processedfor autoradiography using Kodak NTB2 emulsion. The monolayers were stained with hematoxylin, and those cells with a t least five grains were considered to be positive. Two hundred to three hundred cells were counted per cell line. Turnorigenicity Cells transfected with either the pSV2 or pKrev-I plasmids were inoculated subcutaneously into nude mice of BALB/c background from the Laboratory Animal Facility of the Fox Chase Cancer Center. All animals were injected with 2 x 1O6 cells [291. RESULTS Isolation of Krev-1-Transfected Lung Carcinoma Cells The human non-small-cell carcinoma line Calu-6 had two copies of the endogenous Krev-1 gene, but Krev-1 expression was undetectable in northern blots (Figure 1). This cell line was transfected with the pKrev-1 plasmid and 48 h later was selected with G418. After 3 wk of selection, 10 colonies were isolated and grown in vitro. Similarly, two colonies derived from the parental line transfected with the neomycin-resistance gene were isolated and amplified. To further characterize those cells that expressed Krev-I , northern blot analyses were performed with total RNA from all isolated colonies, using cells from passages 10-1 6. From this analysis, three Calu-6-derived lines, i.e., Calu-6 Krev-2, Calu-6 Krev-4, and Calu-6 Krev-7, expressed either moderately or markedly elevated levels of Krev-I mRNA (Figure 1). The level of expression of Krev-I in the Calu-6 Krev-8 line was low; a faint band was seen on northern blots after several days of exposure. No expression of

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Figure 2. Southern blot analysis of the Calu-6 Krev clones. Genomic DNA was extracted from each individual clone, separated by electrophoresis in a l% agarose gel, blotted, and hybridized with a Krev-I cDNA probe.

the Krev-I gene was seen in 65% of the G418-resistant clones, including Calu-6 Krev-9. DNA was extracted from all clones that expressed Krev-I mRNA to characterize the origin of each revertant (Figure 2). These lines were therefore chosen together with the control plasmid transfectants for further studies. The number of copies of Krev-I in each cell line was investigated by dot-blot analysis. Densitometric evaluation of the dot blots revealed that transfectant cells contained two to five copies of the Krev-I gene. All clones derived from Calu-6, as well as the parental line, showed an extra band not present in the placental control on the Southern blot (Figure 2). This was seen in Southern blots using other restriction enzymes and could represent a polymorphism.

a flat or squamous morphology, the control cells were more rounded or ovoid (Figures 3 and 4). Giant cells with large nuclei were also seen more frequently in the Krev-I -transfected cells (Figure 3B). Although the transfected cells were larger and flatter than the control cells, their surface morphology was not very different. Nevertheless, the Krev1 -expressing cells appeared to have fewer and shorter cytoplasmic villi (Figure 4B) than the control cells (Figure 4A). PKC analysis of the Calu-6 cell lines indicated that the Calu-6 Krev-4 and Calu-6 Krev-7 lines had moderately higher PKC activity (Table 1) than the parental cells and the neomycin-transfected-derived clones. Western blot analysis of total immunoreactive PKC was consistent with these results (data not shown). The other two Calu-6 revertant lines assayed (Calu-6 Krev-2 and Calu-6 Krev-8) did not have PKC levels significantly different from controls transfected with the vector only. Approximately one third of the cells incorporated tritiated thymidine in a pulse-label experiment. No significant differences were seen between the labeling indexes of the control and the transfected cells (Table 1 ).

In Vitro Characteristics of Krev-1 Transfectants

Tumorigenicity of Krev-I -Transfected Cells

The morphology of the transfected cells was markedly different from that of either the parental or the control transfected cells. Whereas most Krev-transfected cells had

Subcutaneous tumorigenicity in nude mice was investigated in all transfectants expressing Krev-I mRNA. Table 1 shows the in vivo growth of Calu-6 transfected cells in

Figure 1. Northern blot analysis of the Calu-6 Krev transfectant clones. Fifteen micrograms of total RNA was electrophoresed in a 1% agarose-2.2 M formaldehyde gel. After it was blotted to a nylon membrane, the RNA was hybridized to a 1.25-kb Rsal probe specific for the Krev-1 gene. Ethidium bromide staining of the gel is shown in the bottom panel.

KREV- 1 REVERSION OF TUMORlGENlClTY

255

Figure 3. Phase contrast micrograph ( x 140) of (A) Calu-6 parental cell line and (B) the larger and flatter transfectant Calu-6 Krev-7 cells.

nude mice. When 2 x 1 O6 cells/animal were injected subcutaneously in nude mice, a 40-60% reduction in tumor formation was observed in all Calu-6 Krev-I clones (Table 1). The size of the tumors was measured twice weekly during an 8-wk period (Figure 5). The tumors that developed from these cell lines were three to 10 times smaller than

the ones from the control lines carrying the neomycinresistance gene. In addition, the subcutaneous tumors from partially suppressed cells had a tumor histopathology different from that of the control plasmid-transfected Calu-6 cells. Whereas the cells from the latter were basophilic, elongated, and showed no particular epithelial features,

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Figure4. Scanning electromicrographs(x 1900)of (A)Calu-6 cells and (8)C a b 6 Krev-7 cells. Note the flat morphology and short projectionsof the revertant cells in (6) in comparisonwith the more rounded parental cells in (A).

the Krev-I -transfected Calu-6 tumor cells were polyhedral with clear cytoplasm, a central nucleus, and clearly epithelioid features (Figure 6). Several transfectant clones that did not express Krev-1 mRNA were also injected subcutaneously into nude mice and produced neither reductions in tumorigenicity nor morphological changes. DISCUSSION

Transfection experiments using expression vectors carrying the tumor suppressor genes Rb[4-8] and p53 [9-11 I have been reported recently. Changes in morphology were shown in an osteosarcoma cell line, SAOS 2; a retinoblas-

toma cell line, Weri-Rb 27 [4]; and a bladder carcinoma line, HTB 9 [ S ]transfected with a wild-type Rb gene; but no changes were observed in the human prostatic carcinoma cell line DU 145 [6]. Most of the Rb-positive-derived transfectant lines lost their tumorigenic properties [4-81. No changes in morphology were reported after the expression of the wild-type p53 was restored in the human colorectal carcinoma lines SW480 and SW837 [lo], both of which carry a mutated p53 gene. When the osteosarcoma cell line SAOS 2, which lacks both alleles of the p53 gene, was transfected with wild-type p53, enlarged and flattened cells that exhibited a longer doubling time than the parental line were observed [ I I].In addition, after transfection of the human wild-type p53 gene, all these cell lines showed growth suppression in vivo. Conversely, no effect was seen in a less aggressive colon carcinoma cell line, VACO 235, that harbors a wild-type endogenous p53gene [lo]. In the study presented here, we investigated the functional effects of a known Krev-I cDNA expressionconstruct transfected into a human non-small-cell lung carcinoma line, Calu-6, which carries a spontaneously mutated Ki-ras gene. The Krev-1 gene was driven by the SV40 early promoter and was linked to a neomycin-resistance gene as a selectable marker. DNA and RNA analyses demonstrated the uniqueness of each individual clone and the levels of expression of Krev-I mRNA. Approximately one third of the G418-resistant clones isolated expressed a significant amount of Krev-1 mRNA. A low frequency of reversion was seen previously in transfection experiments using this gene [I41 and also the metastasis suppressor gene nm23 [30]. The Calu-6 Krev transfectants had important morphological changes; the cultures had a flat cell phenotype and many giant cells with large nuclei. The revertant clones also showed important changes in their in vivo behavior after xenotransplantation, i.e., a 40-60% decrease in tumorigenicity, a reduction of tumor size, and a longer latency period. Moreover, the tumors produced by subcutaneous inoculation of the Calu-6 Krev revertants into nude mice consisted of polyhedral cells with epithelioid features, whereas the tumor cells from the parental and control cell lines were less differentiated. The expression of Krevdid not affect the in vitro growth rates of the Krev-transfectedclones, because the doubling times of the clones were marginally lower than those of the controls. These results are consistent with the findings of Kitayama et al. [14,15] using Kirsten sarcoma virus-transformed NIH 3T3 cells and HT1080, a human fibrosarcoma cell line harboring activated N-ras genes. An interesting extra band was seen in the Southern blots that has not been observed in the placental DNA. Although we do not have any information on changes in chromosome 1 in Calu-6 cells, a rearrangement cannot be excluded at this time. Nevertheless, similar changes have been seen in other human tumors (data not shown), indicating the possibility of a polymorphism. In C3H 10Tli2 mouse embryo fibroblasts, the biological effect of Krev-I in reversion of ras transformation is

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Table 1. PKC Activity and Turnorigenicity of Calu-6 Krev-1-Transfected Cells Relative PKC

activity Cell line

Labeling index (in vitro)+

f SEM*

100 f 3 100 k 8 86 ? 4 131 2 16 143 f 10 100 ? 4

Calu-6 C a b 6 pSV-2 Calu-6 Krev-2 Calu-6 Krev-4 C a b 6 Krev-7 Calu-6 Krev-8

38

Number of copies of Krev-I*

Tumorigenicity’ 10110

0 0 2 3

35

36 32 34 33

lO/lO 611 1 411 0 6110 5110

5 3

*The mean of three separate experiments c standard error of the mean (SEM). The values were normalized with respect to the parental cell lines. ‘The percentage of cells labeled by pulse labeling with vitiated thymidine. A total of 200-300 cells per line were counted. ‘Copy number of the exogenous Krev-1 gene determined by quantitative radioanalysis. §Thenumber of tumorsinumber of animals injected For tumor-volume data, see Figure 5

accompanied by reversion of PKC levels lowered by an activated Ha-rasgene [31] (Fernandes ER, Ashendel C, manuscript submitted for publication). Calu-6 cells possess an activated Ki-ras gene, and it is reasonable to hypothesize that in these cells PKC levels may be lower than in the cells of origin. As in C3H 10T1/2 cells, it is possible that expression of Krev-I can elevate PKC levels putatively reduced by activation of Ki-{as. Analysis of the PKC levels in the revertants indicated that this occurred in some revertant clones and was correlated with the level of Krev-I expression. Analyses of mRNA are in progress to investigate whether the alteration in levels of PKC results from altered transcriptional regulation. Recent studies have shown the antagonistic effects of Krev-I and c-Ha-ras proteins. An activating mutant of Krev-I is able to interfere in cell determination during the development of the Drosophila eye, in a signal transduction pathway that includes ras p21 [ 18,191. Alternate path-

ways leading to suppression may also exist, as indicated by the existence of suppressed Krev clones with low or unchanged PKC activity, e.g., Calu-6 Krev-2 and Calu-6 Krev-8. Nevertheless, the correlation between Krev-1 antagonism of ras transformation and increased levels of PKC suggests that altered PKC levels may be a valuable biochemical marker for ras transformation and Krev1 reversion. In addition, the dramatic phenotypic changes observed in the Calu-6 Krev clones suggest that Krev-I is implicated in a differentiation program that will induce the cells to acquire epithelioid characteristics. Further experiments using cell lines derived from other tissues are now in progress to test the possibility that different degrees of differentiation or different types of ras mutations influence the suppressiveeffect of Krev-I . If they do, Krev-I protein may play a role in the process of tumor progression in certain cell types, either by competing with the ras effector, or by

-

----C

L

0

5

C6 K rev4 K rev7

Krev8 Krev2 neo

I-

0

2

4

6

10

Weeks

Figure 5.

Kinetics of tumor growth after subcutaneous inoculation into nude mice.

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Figure 6 . Histology of a C a b 6 tumor and a Calu-6 Krev-7 tumor. Note the larger and more epithelioid cells that constitute the subcutaneous tumor induced in nude mice after injec-

tion of the transfectant Calu-6 Krev-7 cell line (B). Calu-6 parental cells gave rise t o a tumor formed by smaller spindle basophilic cells (A). Hematoxylin-eosin staining, x 160.

acting in a negative signal-transduction pathway that is independent of the ras proteins [ 15,161 In this context, it is interesting to note that Ki-ras activation has been associated with poor prognosis in the most aggressive lung adenocarcinomas [22-241 It IS thus feasible that Krev-I could act by neutralizing this effect on progression, thus resulting in a less advanced malignant phenotype Although important regressive changes appear to be induced in Krev-I transfectants, expression or overexpression of the Krev-I gene was not sufficient to completely suppress the malignant phenotype Instability of the transfectants did not seem to account for this Southern analysis of tumors produced by Krev-I -transfected cells showed that even the large tumors produced by inoculation

of unsuppressed Krev-transfected cells retained the exogenous Krev-I gene. The persistent suppression of tumorigenicity in one clone, Calu-6 Krev-8, together with its marginal expression of Krev-I mRNA, is difficult to explain. One possibility is that other genetic alterations were induced by the transfection and culture procedures, and that these resulted in a paradoxical effect. Nevertheless, this report shows that Krev-1 was able to partially suppress the neoplastic phenotype in transfected clones of a malignant human non-small-cell lung carcinoma cell line. Because many events are involved in the initiation and progression of most human cancers, the inability of Krev-I to totally suppress tumorigenicity is not unexpected. Further studies underway in our laboratory transfecting multiple genes

KREV- 1 REVERSION OF TUMORlGENlClTY

with suppressive activities could eventually result in complete suppression. ACKNOWLEDGMENTS

This work was supported by NIH grants CA-44981, CA-53713, and CA-06927 and by an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center. Received May 28, 1992; revised August 7, 1992, accepted August 11. 1992

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