Spontaneous transformation of an immortalized hepatocyte cell line: potential role of a nuclear protease

Cancer Letters 213 (2004) 39–48 www.elsevier.com/locate/canlet

Spontaneous transformation of an immortalized hepatocyte cell line: potential role of a nuclear protease David A. Drubin, Gary A. Clawson* Departments of Pathology and Biochemistry and Molecular Biology, The Jake Gittlen Cancer Research Institute, H059, Hershey Medical Center, The Pennsylvania State University, 500 University Drive, Hershey, PA 17033, USA Received 30 December 2003; received in revised form 23 March 2004; accepted 24 March 2004

Abstract In this study, we utilized an in vitro model of spontaneous transformation/progression, an SV40 large T antigen-immortalized rat hepatocyte cell line (designated CWSV14) that is very weakly tumorigenic at low-passage, but acquires a transformed phenotype upon extended passage in cell culture. Here we show that this mid-passage transformation is accompanied by development of aneuploidy and disorganization of the actin cytoskeleton, concomitant with a large increase in a chymotrypsinlike nuclear protease activity which we have previously implicated in chemical transformation of fibroblasts and rastransformation of hepatocytes. Passage of the CWSV14 cells with AAPFcmk, a relatively selective inhibitor of the nuclear protease activity, abrogates the acquisition of the transformed phenotype and prevents the changes in the actin cytoskeleton. We hypothesize that the nuclear protease may play a role in initiating development of genomic instability, paralleling the archetypical role of proteases in paradigms such as the SOS-type responses in bacteria and yeast. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Carcinogenesis; Protease inhibitors; Nuclear protease; Nucleus; SV40

1. Introduction Over the years, a select group of protease inhibitors (PIs) have consistently exhibited effective anticarcinogenic activity in a wide variety of model systems (for review, see Ref. [1]). The laboratory of Troll first described this phenomenon in a model of Abbreviations: PI, protease inhibitor; BS, Bloom’s syndrome; NS, nuclear scaffold; NSPA, nuclear scaffold-associated protease activity; cmk, chloromethyl ketone; amc, 7-amino-4-methyl coumarin. * Corresponding author. Tel.: þ1-717-531-5632; fax: þ 1-717531-5298. E-mail address: [email protected] (G.A. Clawson).

mouse skin carcinogenesis in which an inhibitor of chymotrypsin-like proteases and two inhibitors of trypsin-like activity were each individually able to suppress the tumorigenesis initiated by 7,12dimethylbenz(a)anthracene and promoted by phorbol esters [2]. Many other in vivo systems of carcinogenesis mimic this phenomenon in different organ systems (lung, colon, mammary gland, liver, oral mucosa, lymphatics, and esophagus), in different species (rat and hamster), with different types of PIs (antipain, leupeptin, and the Bowman – Birk Inhibitor), and with different carcinogenic agents (radiation and various chemical carcinogens) (for review, see Ref. [1]). Various in vitro models of transformation,

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.045

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particularly using either radiation or 3-methylcholanthrene as carcinogens, show parallel anticarcinogenic effects by those PIs used in vivo (for review, see Ref. [1]). In addition, PIs have also displayed pronounced effects on genetic susceptibilities to cancer, significantly suppressing the development of tumors in Min mice [3], an autosomal-dominant inheritance of a predisposition to multiple intestinal neoplasms, the onset of primary hepatocellular carcinomas observed in the inbred C3H mouse strain [4], as well as the chromosome instability in cells from patients with the cancer predisposition disorder Bloom’s syndrome (BS) [5]. Most studies showing robust chemopreventive activity of PIs have employed inhibitors of chymotrypsin-like proteases [1,6]. While the impressive anticarcinogenic activities associated with PIs have been described for several decades, biological mechanisms have remained enigmatic. Furthermore, although multiple candidate protease targets have been described [1,7 – 10], they have remained ill-defined [11]. One interpretation that can be drawn from the similar effects of the anticarcinogenic PIs in various model systems is that there is a common pathway in the process of carcinogenesis regulated by a protease (or set of proteases) that is activated independent of either carcinogen or cell type. Thus with this view, characterizing the protease target(s) involved in one system could lead to the unveiling of mechanisms for many. Our past investigations [12 – 14] have described a specific proteolytic activity, associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts, that is potentially involved in carcinogenesis. The nuclear scaffold-associated protease activity (NSPA) was first identified by its preferential digestion of the nuclear lamins A/C [14], resulting in a truncated lamin species lacking the nuclear localization signal. Both in vitro and in vivo, the NSPA is Ca2þ regulated [12,15], and it displays a chymotrypsin-like substrate preference [12]. The NSPA has also been found to be elevated in rat livers exposed to various chemical carcinogens [14], and to closely mirror the mitotic index in liver regeneration [16]. All together, these three characteristics of the NSPA: the unique juxtaposition of the NSPA with the NS (and thus DNA and DNA-binding proteins), its chymotrypsin-like

substrate preference, and its elevation with carcinogen exposure and cell replication, suggest that NSPA is an intriguing potential target for anticarcinogenic PIs. We identified a chloromethyl ketone (cmk) inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone (AAPFcmk), which exhibits a KI of 56 nmol/l for the NSPA in 10T1/2 mouse embryo fibroblasts [12] and, at appropriate concentrations, is selective for protease inhibition in the NS fraction [12,17]. The inhibitor AAPFcmk displayed striking effects, significantly inhibiting foci formation induced by 3-methylcholanthrene in an in vitro fibroblast transformation assay even at femtomolar concentrations [13], while protease inhibition was relatively specific for the NS fraction [12]. Further, AAPFcmk inhibited the growth of a ras-transformed, SV40 large T antigen-immortalized hepatocyte cell line (NR4) without affecting the growth of the parental large T antigen-immortalized cell line [17,18]. This AAPFcmk-induced growth arrest was accompanied by a notable reorganization of the actin cytoskeleton in the ras-transformed cells [17], and localization of the inhibitor predominantly to the nucleus and nuclear periphery was demonstrated [17]. Taken collectively, anticarcinogenic properties of NSPA-selective AAPF cmk treatment have been demonstrated in two independent models. For this study we have made use of a SV40 large T antigen-immortalized rat hepatocyte cell line [19 – 22], termed CWSV14, to investigate the role of the NSPA in an in vitro model of spontaneous transformation and thereby to further establish a common role for the NSPA in a process of carcinogenesis that is independent of the causative factor. CWSV14 cells are one clone in a series of SV40 large T antigen-immortalized rat hepatocytes (the CWSV series), which express albumin and other liver specific proteins. CWSV14 cells express albumin at 70% of levels in rat liver, but lose albumin expression and begin to express a-fetoprotein upon extended passage (after about 30– 40 passages) in culture [21]. Our interest in the CWSV14 model lies in its transition from a weakly tumorigenic phenotype to a strongly tumorigenic phenotype during this mid-passage period when grown in culture [21], thus providing a reproducible system in which to observe a spontaneous transformation (or perhaps more appropriately a ‘progression’) event. In the present study we assess various aspects of this spontaneous increase in tumorigenic potential in

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the CWSV14 model, and examine effects of AAPFcmk on the transformation. In so doing, we provide a characterization of the anticarcinogenic PI phenomenon in the relatively unexplored area of in vitro spontaneous transformation.

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periodically read on the fluorometer in capillary tubes (Amersham Biosciences, Piscataway, NJ, USA). Activity is expressed as the linear average change in fluorescence units per minute. 2.3. Soft agar assays

2. Materials and methods 2.1. CWSV14 cell culture These studies used the CWSV14 cell line [19 – 22] which was maintained as previously described [19], with the substitution of DMEM-F12 base media (Gibco-Invitrogen, Carlsbad, CA, USA) for the RPMI 1640 base of the RPCD chemically defined media (here referred to as DFCD). AAPFcmk (Enzyme Systems Products-MP Biomedicals, Livermore, CA, USA) was solubilized in DMSO (Sigma, St Louis, MO, USA) at a stock concentration of 20 mM and was applied to cells in the media every two days concomitant with the addition of fresh medium. As a solvent control, an equivalent volume of fresh DMSO was applied to parallel plates. 2.2. Protease assays of nuclear scaffold preparations Nuclei and NS were prepared as previously described [13]. Once NS preparations were obtained from the CWSV14 cells at various passages, protein concentration was determined via the method of Lowry [23]. Five micrograms of NS protein for each experimental group was then assayed for chymotrypsin-like protease activity using another commercially available chymotrypsin substrate coupled to 7-amino4-methyl coumarin (amc), succinyl-Leu-Leu-Val-Tyramc, (LLVYamc) (Enzyme Systems Products-MP Biomedicals). Upon cleavage, the released amc fluoresces and is detected at an emission wavelength of 460 nm (excitation wavelength is 380 nm). For measuring fluorescence, we used a TKO 100 Hoefer DNA Fluorometer (Hoefer Scientific Instruments, San Francisco, CA, USA). The protease assays were conducted in 100 ml reactions: They included 1 mM (final concentration) LLVYamc, 10 ml of DMSO, and 5 mg of NS protein in 90 ml TKMC (5 mM MgCl2, 25 KCl, 50 mM Tris pH 7.4, 10 mM CaCl2). Reactions were incubated at 37 8C and fluorescence was

The soft agar assay was employed to test the transformation of CWSV14 cells, based on the ability to grow with anchorage independence. A stock of 1.2% Seakem GTG agarose and 2 £ DFCD with 3% active FBS (HyClone, Logan, UT, USA) was made for the preparation of a 0.4% bottom agar and 0.2% top agar. Media base without phenol red was used to make possible the subsequent counting of macroscopic transformed colonies on the autocounter. After culturing the CWSV14 cells from relatively low passage (pass 27– 30) to high passage (pass 50– 55) as described above, cells were trypsinized and counted with a hemocytometer (Fisher Scientific, Pittsburgh, PA, USA) to determine appropriate volumes for cell seeding. The soft agar assays were established in 60 mm petri dishes with 5 ml of bottom agar and 2 ml of top agar containing 1 £ 105 CWSV14 cells of appropriate treatment groups. Appropriate amounts of DMSO or AAPFcmk were also added to both the top and bottom agar. Three plates were prepared for each soft agar colony count day planned per treatment group, and they were incubated at 37 8C with 5% CO2 until counted. Plates were stained with 2 ml of 1 mg/ml p-iodonitrotetrazolium for 24 h, and then counted on a Dynatech Lab autocounter, set to count colonies 0.2 mm in diameter or greater. The average of the three plates for each treatment group was taken for a particular count day. If not yet counted, cells on the agar plates were fed with an additional 2 ml of top agar on the fifth day after initial plating, and every three days after the fifth day, just 2 ml of 2 £ DFCD was added. All media additions to the soft agar plates included appropriate treatments of AAPFcmk or DMSO. 2.4. Flow cytometry and DNA content analysis Flow cytometry of propidium iodide stained cells was performed as previously described [17]. To determine the CWSV14 DNA index, rat peripheral blood lymphocytes (PBLs) were isolated as diploid

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controls. Briefly, 5 ml of peripheral rat blood was drawn into syringes coated with EDTA to prevent coagulation. This was mixed with 5 ml of PBS, and the mixture was placed on a 5 ml Lympholyte (Cedarlane, Hornby, Ont., Canada) cushion for centrifugation-separation of blood components (25 min at 1000 £ g). The lymphocytes were removed from the interphase between the media and lympholyte layer, and any contaminating red blood cells were subsequently lysed with a short incubation in 2 ml ACK lysing buffer (Bio-Whittaker, Walkersville, MD, USA). Purified PBLs were then spiked in with the collected CWSV14 cells and stained for flow cytometry as described [17]. Significance was established at P # 0:05 by use of a two-tailed Student’s t-test. 2.5. Actin staining Actin staining was performed directly in the tissue culture plates. The adhered cells were first washed three times with 1 £ PBS pH 7.5. Next, cells were fixed with 3% neutral buffered formalin (in H2O) for 45 min at room temperature and then washed again three times with 1 £ PBS. Permeabilization of the cells was achieved with an incubation of 20 min at room temperature in 1% Igepal-CA-630 (Sigma) prepared in 1 £ PBS. Cells were then washed three times with 1 £ PBS. The actin cytoskeleton was then stained with rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA), diluted 1:100 in 1 £ PBS, for 30 min at 37 8C, and the cells were subsequently washed three times with 1 £ PBS. Counterstaining of the cell nuclei was carried out by incubating cells with 300 nM of DAPI (Molecular Probes), in 1 £ PBS, for 5 min at room temperature. Cells were given a final 1 £ PBS wash and mounted with coverslips using an antifade kit (Molecular Probes).

3. Results Historically, PIs have been associated with anticarcinogenic activities in a variety of model systems. The biological mechanisms underlying these effects, however, have remained undefined. The wide-variety of systems in which these anticarcinogenic effects are

observed suggests a common mechanism of carcinogenesis involving proteases. In order to determine whether the NSPA, previously identified by our laboratory and demonstrated to be involved in chemical carcinogenesis, may represent a protease targeted by the anticarcinogenic PIs and involved in a potentially common process of carcinogenesis, we further characterized the NSPA in an in vitro model of spontaneous transformation, an arena insufficiently explored in previous studies of protease inhibitors and carcinogenesis. The hepatocyte-derived CWSV14 cell line [19 –22] was chosen as a model system to further study the role of the NSPA in carcinogenesis because of its characteristic spontaneous transformation upon continued passage in culture [21]. Here, experiments were started with relatively low-passage aliquots (pass 30 or lower), when the cells display low tumorigenic potential. They were grown in culture through to the high-passage stage (pass 50 and above), when the cells exhibit high tumorigenicity. Cells at both low and high passage were analyzed for growth in soft agar to determine anchorage-independent growth. As expected, low-passage CWSV14 cells showed little ability to grow in an anchorageindependent manner, while at high-passage colony formation in soft agar was greatly increased (data not shown). This acquisition of anchorageindependent growth parallels previous work demonstrating acquisition of tumorigenicity for CWSV14 cells in vivo [21]. To determine if this phenomenon of increased tumorigenic potential relates to the NSPA, CWSV14 cells were harvested at low, middle, and high-passage stages, and the NSPA was measured. Concomitant with the acquisition of anchorage-independent growth described above, the cells exhibited a 300% increase in the NSPA, and a similar elevation in NSPA was also observed in a cell line derived from a tumor which developed after transplantation of CWSV14 cells in syngeneic rats (CWSV14T1) (Table 1). As the development of genomic instability and aneuploidy is a common characteristic of transformed cells [24,25], we also wanted to assess whether any DNA content changes occurred during transformation in this model. Flow cytometric analyses of cells stained for DNA content at the various passage stages showed that the cell populations did develop

D.A. Drubin, G.A. Clawson / Cancer Letters 213 (2004) 39–48 Table 1 Nuclear scaffold-associated protease activity in CWSV14 cells with passage in culture Cell line

Nuclear scaffold chymotrypsinlike protease activity (mean units/min ^ SE)

CWSV14 low-passage (,pass 30 or less) CWSV14 mid-passage (pass 35–45) CWSV14 high-passage ðpass . 50Þ CWSV14T1

0.115 ^ 0.093 0.470 ^ 0.030* 0.487 ^ 0.046* 0.483 ^ 0.112**

*P , 0:0004 compared to CWSV14 low-passage cells; **P , 0:002 compared to CWSV14 low-passage cells.

aneuploidy, exhibiting an increase in DNA content at the same passage interval where transformation and increases in NSPA were observed (Fig. 1). The CWSV14 cells are tetraploid (DNA index of 2)

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at low passage (a property of all CWSV cells lines we have examined), and they develop aneuploidy during the transition through the mid-passage stage (Fig. 1). Aneuploidy became significant compared to low passage cells (at P # 0:05) as the cells reached the high-passage stage. Assuming the DNA content was increasing progressively during culturing, the DNA index of the high-passage cells was also significantly increased compared to cells in the mid-passage stage (at P , 0:05). Karyotypic analyses were not performed; however, we would predict the accumulating karyotypic changes are random in nature. We next wished to determine if the increased NSPA is mechanistically involved in development of transformation in this model. CWSV14 cells were again cultured from low-passage to the highpassage stage, either: (A) without treatment; (B) in the presence of 0.125% DMSO (solvent control); or (C) with 25 mM AAPFcmk, a relatively specific, irreversible inhibitor of the NSPA. Concentrations

Fig. 1. Flow cytometry of CWSV14 cells with continuous culturing. To assess whether aneuploidy may develop concomitant with spontaneous transformation in the CWSV14 model, cells were continuously passaged in culture (as indicated), and were then harvested and stained with propidium iodide. DNA content was measured via flow cytometry. In (A) a positive, non-integral shift in DNA content as the cells were cultured from low-passage (pass 30 and under) to about the mid-passage stage (pass 40 and above) is graphically demonstrated. The staining shows the cells in normal logarithmic growth with cells distributed in all phases of the cell cycle. In (B), the table shows the quantitative DNA index, which is the ratio of mean CWSV14 G1 DNA content to normal diploid rat PBL DNA content. CWSV14 cells are actually tetraploid in culture (DNA index of 2). With continued passaged, the non-integral increase in DNA-index is significant at P # 0:05 (two-tailed Student’s t-test) at about pass 50 and above (high-passage). The increase in DNA index is also significant from mid-passage to high passage at P , 0:05 (one-tailed Student’s t-test), assuming a progressive increase in DNA content as cells were passaged.

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Even more significantly, when compared to cells treated with the same concentration of solvent alone (0.125% DMSO), the inhibition of colony formation was 94%. The enhancement of CWSV14 anchorage independent growth (about twofold) observed with the treatment of 0.125% DMSO is of interest, since DMSO has been found to be mutagenic in bacterial tester strains [26,27]. Once tumorigenic, however, neither AAPFcmk nor DMSO had an effect on the ability of CWSV14 cells to form colonies in soft agar (data not shown).

4. Discussion Fig. 2. CWSV14 actin organization with continuous culturing. CWSV14 cells were grown from low-passage (under pass 30) to high-passage ($pass 50), either in the presence of 25 mM AAPFcmk (A and B) or solvent (0.125% DMSO) alone (C and D). Panels shown are representative images from rhodamine-phalloidin stained CWSV14 cells at about pass 50. Those cells grown in the presence of 25 mM AAPFcmk (A and B) have a more defined and organized cytoskeleton than those grown with the solvent control alone (C and D), which exhibit few cytoskeletal fibers.

up to 50 mM AAPFcmk had no effect on the growth rate of CWSV14 cells (data not shown), similar to previously reported results with CWSV1 cells [17], indicating that any growth-related results that follow are not due to effects on growth rate. As ras-transformed variants of the CWSV series of cells lines characteristically show disorganization of the actin cytoskeleton [17], we assessed the condition of the actin organization as a marker for transformation in the CWSV14 model. Actin staining of the CWSV14 cells demonstrated that when they were passaged in the presence of AAPFcmk, CWSV14 cells maintained a relatively organized cytoskeleton, compared to cells in the solvent only control (Fig. 2). This is reminiscent of the ‘actin-organizing’ effect we observed with 25 mM AAPFcmk treatment of rastransformed hepatocytes [17]. In soft agar colony assays, which we demonstrated here to parallel in vivo measures of tumorigenicity in the CWSV14 model, inclusion of AAPFcmk also prevented the characteristic acquisition of anchorageindependent growth with passage. Cells treated with 25 mM AAPFcmk exhibited 86% fewer colonies by day 7 after plating than did untreated cells (Fig. 3).

Collectively, these results suggest that the NSPA may have an important role in the spontaneous transformation observed upon extended culturing of

Fig. 3. Soft agar assay of CWSV14 cells cultured in the presence of AAPFcmk. CWSV14 cells were cultured from passage 30 to passage 51 (low-passage to high-passage) either: (A) in the presence of 25 mM AAPFcmk; (B) with solvent alone (0.125% DMSO); or (C) with no treatment. They were then assessed for anchorageindependent growth capacity via the soft agar assay in the presence of AAPFcmk, with solvent alone (0.125% DMSO) or with no treatment. Data shown are the number of p-iodonitrotetrazoliumstained colonies of 0.2 mm in diameter or greater that grew in soft agar 7 days after seeding. The values obtained are from triplicate readings from three plates of each treatment condition. Similar trends were also observed with continuous treatment of 50 mM AAPFcmk and 0.25% DMSO (data not shown). The difference in colony number between the AAPFcmk treatment group and either the solvent control group, or the untreated group is significant at P , 0:02 (one-tailed Student’s t-test). Furthermore the difference in colony number between the DMSO treated group and the no treatment group is significant at P , 0:05 (two-tailed Student’s t-test).

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this SV40 large T antigen-immortalized CWSV14 cell line. This is the first characterization of a nuclear protease activity in a model of in vitro spontaneous transformation. Spontaneous transformation, aneuploidy, and elevation of the NSPA all occur concurrently in the CWSV14 model, and treatment with a relatively specific NSPA inhibitor abrogated the acquisition of the ability to grow in soft agar. Even taking into account the caveat that the inhibitor is relatively specific for the NSPA and effects could potentially reflect inhibitory activity towards other chymotrypsin-like protease activity in the cell (unlikely, though, given the past demonstrations of selectivity for the NS compartment at the appropriate concentrations [12,17] and the correlative elevations of the NSPA in this study and in chemical carcinogenesis [14]), these results extend previous work demonstrating anticarcinogenic properties of PIs targeted to chymotrypsin-like proteases in models of chemical, physical, and genetically predisposed carcinogenesis [1] by showing PI-mediated suppression of an in vitro spontaneous transformation event involving the expression of a viral oncoprotein. The large T antigen [28] is an oncoprotein of SV40, a DNA tumor virus which has the ability to transform hamster, mouse, and rat cells in vitro. While hamster cells do not need to be passaged in culture to be transformed by SV40 [29], mouse [30 –33] and rat cells [21] require prolonged culturing. In the rat CWSV14 cell line we observe an elevation in the NSPA during extended culturing that likely reflects a cellular response to the continuous expression of large T-antigen, which could represent a mechanism behind SV40-mediated transformation. As the NSPA is also found elevated in rat livers in response to chemical carcinogen exposure [14], it is reasonable to propose that a similar process may be activated in these two different models of carcinogenesis. It is also reasonable to propose that, given the inhibition of transformation by the relatively specific inhibitor (which has shown a high selectivity for the NS compartment at the concentration used [17]) in this study and previously in a model of chemical carcinogenesis [13], the NSPA up-regulation is necessary for progression of the carcinogenic cascade in these two systems. The lack of antitumorigenic effects of AAPFcmk on CWSV14 cells that have already been transformed further suggests that whatever process

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that AAPFcmk interrupts is required in carcinogenesis only for tumorigenicity to arise. At this point, another event must occur which causes the cells to be refractory to the anticarcinogenic effects of AAPFcmk. This is in agreement with conclusions drawn from earlier mouse 10T1/2 fibroblast carcinogenesis studies demonstrating no effect of PIs on cells already transformed [34 – 36]. Given our results and the collection of data on the anticarcinogenic activity of many PIs in myriad systems, it seems clear that a protease (perhaps multiple proteases) in some way plays a key, common role in carcinogenesis. We propose that the NSPA is a key target of the anticarcinogenic PIs. Mechanistically, our data temporally links development of genomic instability (aneuploidy) with a rise in the NSPA. This is of considerable interest since inhibitors of chymotrypsin-like protease activity have previously been shown to suppress chromosomal instability: inhibiting UV-induced mutation and gene conversion events in yeast [37] and chromosomal abnormalities and aberrant recombination in fibroblasts from patients with BS [5], an autosomal recessive chromosomal instability disorder that predisposes the patient to a wide variety of neoplasms [38]. While UV irradiation induces the recombination in the yeast model, chromosomal instability in BS is caused by the disruption of the BLM gene which encodes a DNA helicase involved in DNA repair functions and suppression of illegitimate recombination at stalled replication forks [39]. These represent two vastly different causes of chromosomal instability, yet PIs suppress the instability in both cases. The temporal correlation of the increased NSPA with the development of aneuploidy in the CWSV14 model conceptually supports a third system in which chromosomal instability is associated with a protease activity. This also reinforces the previous suggestion (based on the common inhibition by chymotrypsinlike protease inhibitors) of a common mechanistic link (via a protease activity) between the radiationinduced recombination in yeast, the chromosomally unstable cellular phenotype of BS, and the general induction of malignancy (which involves genomic instability) in mammalian cells [1]. Whatever the details of the mechanism, it is becoming more established that the effects of the anticarcinogenic PIs are likely the result of inhibition of a protease(s)

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tightly involved in the regulation of genomic instability. The idea of a protease activity regulating a cellular state of genomic instability, however, is not a new concept. The SOS system is a well-established survival response [40 – 42] to DNA damage in bacteria, which is triggered by the induction of a protease activity and is regulated at different levels by proteases [43]. In fact, certain PIs can inhibit the functions of the SOS response [44]. The SOS response is initiated with the acquisition of a coprotease activity by RecA after it forms a nucleoprotein filament on single-stranded DNA regions, which arise when replication forks stall at damaged DNA template bases. The nucleoprotein filament form of RecA, RecA*, then participates in the autocleavage reaction of the multioperon repressor, LexA [45], allowing the transcription of 43 SOSresponsive genes. The survival and adaptive capacity that ultimately results from activation of the SOS response requires the error-prone component of the SOS response and is accompanied by mutation [42,46], causing an increase in genetic variation among the surviving population. The mutagenesis associated with the error-prone arm of the SOS response is tightly regulated. One way in which mutagenesis is controlled downstream of SOS initiation is by protein– protein interactions and the selective proteolytic degradation of specific protein complexes [43], and one protease involved in this protein complex degradation, ClpXP, is actually required for mutagenesis in response to UV radiation in E. coli [43], demonstrating the degree to which a single protease can be involved in mutagenesis and what can happen when a specific protease activity is eliminated. This regulated mutagenesis system exemplifies the concept posited by Wintersberger that portrays mutagenic agents as carcinogens in such a way that the mutations leading to cancer in multicellular organisms are not directly caused by the mutagenic agents themselves, but arise as a result of a programmed genomic mobilization that ensues in response to the initial DNA damage caused by the mutagenic agents [37]. Our working hypothesis is that the NSPA is involved in the induction or regulation of a response of the SOS archetype in mammalian cells, a response to DNA damage (or similar cellular conditions normally created by DNA

damage) that leads to a cellular program of survival by genomic instability. A response system in eukaryotes with the same modus operandi as the bacterial SOS response has not been found [47]. This is perhaps not surprising, given the major differences of genome organization, replication mechanisms, and overall DNA metabolism between prokaryotes and eukaryotes. If a system analogous to the SOS response does exist in eukaryotes, it would presumably work quite differently. Given the DNA compartmentalization within the nucleus in eukaryotic cells, nuclear structure and function could play an important role in such a pathway. In fact, the NS plays a vital role in nuclear function, providing a three-dimensional framework upon which nucleic acid metabolism occurs [48]. DNA replication, which is tightly linked to DNA repair and recombination, takes place attached to the NS in discrete nuclear regions [48 – 52], and the transcriptional state of chromatin is also highly dependent upon the structural organization within the nucleus [48]. It is easy then to imagine how a protease associated with the NS could play a role in all these processes, either through a form of signaling or through structural modulation. Whatever the mechanism might be, it is important to note that the genomes of eukaryotic cells do become unstable in response to stresses such as radiation [37,53], and do gain survival and growth advantages, manifesting as cancer is metazoans. So, although what is good for the survival of the single cell is not necessarily good for the survival of the whole organism, in light of the results produced by the anticarcinogenic PIs (such as AAPFcmk), the existence of a eukaryotic representation of the bacterial SOS response archetype seems quite possible. The novel NSPA, which has now been demonstrated to have a role in several different models of carcinogenesis, including a viral oncogene-related spontaneous transformation event described in this study, and has a unique juxtaposition with DNA and nuclear structure, may be a key to defining a potential eukaryotic SOS-type response and understanding better the early stages of carcinogenesis. While currently undefined, the protein(s) behind the NSPA is being actively pursued by our laboratory. We have obtained some sequence information in this regard and presently are in the process of cloning and better characterizing this novel serine protease.

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Acknowledgements We thank Dr Harriet Isom for kindly providing the CWSV14 cell line. We also thank Shelley Gestl, Thomas Miller, and Mary Pickering for technical support, and Nate Shaeffer of the flow cytometry core facility. Portions of this work were supported by NIH/NCI (CA21145 and CA40145), and by funds from the Jake Gittlen Cancer Research Institute.

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