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Carcinogenesis, Vol. 23, No. 3, 521-528, March 2002
© 2002 Oxford University Press

CARCINOGENESIS

Mechanism of lovastatin-induced apoptosis in intestinal epithelial cells

Banke Agarwal1,4,*, Balazs Halmos1,*, Aleksander S. Feoktistov1, Petr Protiva1, William G. Ramey2, Ming Chen3, Charalabos Pothoulakis3, J.Thomas Lamont3 and Peter R. Holt1,5

1 Department of Medicine and
2 Department of Surgery, St Luke's–Roosevelt Hospital Center, College of Physicians and Surgeons, Columbia University, New York, NY, 10025 and
3 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 and
4 Department of Gastrointestinal Medicine and Nutrition, MD Anderson Cancer Center, Houston, TX, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 References
 
We earlier showed that lovastatin potentiated the chemopreventive effects of sulindac against colon neoplasia in a rodent model and augments apoptosis induced by 5-FU and cisplatin in human colon cancer cells. In the present study, we investigated effects of lovastatin in spontaneously immortalized rat intestinal epithelial cells, IEC-18 and their K-ras transformed clones. Lovastatin induced morphologic changes (cell rounding and detachment) and apoptosis that were not influenced by K-ras mutations, but were prevented by geranylgeranyl-pyrophosphate or by mevalonate. Clostridium difficile toxin B, which directly inactivates rho, induced similar morphologic changes and apoptosis. Cycloheximide prevented these effects of lovastatin, but not C. difficile toxin B. Lovastatin decreased the amounts of membrane bound rhoA and rhoB. Cycloheximide and geranylgeranylpyrophosphate prevented lovastatin induced morphologic changes and apoptosis but did not inhibit lovastatin-induced changes in membrane translocation of rho. Our data suggest that lovastatin induces morphologic changes and apoptosis by inhibiting geranylgeranylation of small GTPases of the rho family and thereby inactivating them. Restoration of membrane translocation of rho is not necessary for preventing lovastatin-induced morphologic changes or apoptosis.

Abbreviations: CHX, cycloheximide; FPP, farnesylpyrophosphate; GDI, G-protein dissociation inhibitor; GGPP, geranylgeranyl-pyrophosphate.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 References
 
Lovastatin, an HMG-CoA reductase inhibitor, has profound cellular effects, including inhibition of proliferation, induction of apoptosis and inhibition of stress fiber formation (1–9). Lovastatin is used clinically to reduce serum cholesterol levels and also lowers the incidence of myocardial infarction (10) and ischemic cerebral strokes (11), slows the progression of chronic renal failure (12) and increases bone density in patients with osteoporosis (13). In two clinical trials that evaluated coronary events in patients taking HRIs, there was a 43% and 19% decrease in the number of new cases of colon cancer diagnosed over a 5-year follow-up period in patients taking pravastatin and simvastatin respectively (14,15). We earlier demonstrated that lovastatin augments sulindac-induced apoptosis in colon cancer cells and potentiates the chemopreventive effect of sulindac upon colon cancer in an animal model (16). We also found that lovastatin increases apoptosis induced by the standard chemotherapeutic agents 5-fluorouracil and cisplatin in colon cancer cells (17).

Lovastatin inhibits the synthesis of isoprenoids (such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate) that are byproducts of the cholesterol synthetic pathway. Post-translational isoprenylation, through the activity of the enzymes farnesyl transferase and geranylgeranyltransferase I and II (18,19) is important in determining membrane localization and function of many cellular proteins including small GTPases like ras, rho and rab (20). Since ras mutations are frequent in tumors (21) and because farnesylation is critical for ras function, early studies of lovastatin action in tumor cells focused on its ability to inhibit ras farnesylation (22), but it is now believed that this may not be the main mechanism by which lovastatin inhibits proliferation and induces apoptosis (23). Indeed, inhibition of geranylgeranylation of rho family proteins is now under consideration as a possible critical mechanism of lovastatin effects. Rho proteins are a family of small GTPases belonging to the Ras superfamily that are involved in the maintenance of cellular architecture, inter- and intracellular signaling, proliferation and apoptosis (24–30). Rho proteins are downstream effectors of the ras-pathway and participate in integrin, cadherin and growth factor signaling (31–33). The rho family of proteins also are targets of several important bacterial toxins, including toxins A and B of Clostridium difficile which glucosylate rho, cdc42 and rac (34,35), resulting in their inactivation and causing breakdown of the actin cytoskeleton, cellular detachment and apoptosis (36,37).

After activation, rho proteins translocate to cell membranes in order to carry out their function (38). Translocation of newly synthesized rho from cytosol to plasma membrane occurs during transition of cells from G1 to S phase (32). However the role of this translocation in apoptosis induced by lovastatin or the farnesyl transferase inhibitors (FTIs) has not been studied.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 References
 
Materials
The IEC-18 parental cell line, vector clone C1 and the IEC-18-R1, R4, R8 and R10 ras transformed clones (39) were kindly provided by Prof. I.B.Weinstein of the Columbia Presbyterian Cancer Center, Columbia University, New York, NY. The R10 cells have been characterized as lacking any functional p53 protein as they have a mutation on codon 161 and the other allele is silent (39). Lovastatin was kindly provided by Merck Laboratories, Rahway, NJ. Geranylgeranylpyrophosphate, farnesylpyrophosphate, mevalonate and cycloheximide were purchased from Sigma, St Louis, MO. C. difficile toxin B was purified from VPI 10463 strain (ATCC) culture supernatant as previously reported (40).

Cell culture
IEC-18, C1, R1, R4, R8 and R10 cells were maintained in Dulbecco's modified Eagle medium (Gibco-BRL, Grand Island, NY) with 10% fetal bovine serum (Gibco) in an atmosphere of 95% air and 5% CO2 at 37°C. Cells were grown to 70% confluence prior to treatment with lovastatin or other compounds. All cultures were passaged weekly and fed three times a week. In all experiments, compounds to be studied were added to the medium at the start of the experiment (unless stated otherwise).

Flow cytometry
Flow cytometry was used to quantitate apoptotic cells. Cells were analyzed on a FACSORT flow cytometer (Becton-Dickinson, San Jose, CA) following staining using a commercially available Apo-BrdU kit (Phoenix Flow Systems, San Diego, CA).

Sub-diploid peak analysis
Cells were trypsinized, washed with PBS twice and kept in 70% ethanol overnight at –4°C. Propidium iodide/RNase was added, cells were incubated for 30 min at room temperature and then were analyzed by flow cytometry. The data were plotted on FL2-H histograms and the number of sub-G1 cells were counted as apoptotic cells.

Apo-BrdU (TUNEL) staining
DNA strand breaks in apoptotic cells were detected by incorporation of fluorescein-labeled deoxyuridine triphosphate into fragmented DNA by terminal deoxynucleotidyl transferase. Briefly, cells were trypsinized, were washed twice with PBS, then were resuspended in 5 ml of 1% paraformaldehyde for 15 min at 0°C, washed with PBS and kept overnight in 70% ethanol at –4°C. Cells were washed with washing buffer twice, were incubated with BrdU labeling reagents overnight, were washed twice with rinsing buffer and were incubated with fluorescein tagged antibody to BrdU for 30 min. Propidium iodide with RNAase then was added and the cells were analyzed by flow cytometry after 30 min. The data were plotted on a dot plot -FL2-A versus FL2-W and a singlet gate was applied. These gated cells then were plotted on dot plot FL1-H(log) versus FL2-A(lin) and the cells which stained with BrdU were counted as apoptotic.

Sub-cellular fractionation and western blotting
Cells (both floating and adherent) were collected into PBS/1 mM PMSF and were washed in PBS/1 mM PMSF twice. Cells were resuspended in 1.5 ml of 0.33 M sucrose in TEM-PI Buffer (20 mM Tris pH 7.4, 1 mM EGTA, 5 mM MgSO4 and protease inhibitors) and were homogenized using #2 Dounce homogenizer. Nuclei and insoluble material were removed by centrifugation at 15 000 g for 15 min at 4°C. Membranes and cytosol were separated by centrifugation at 150 000 g for 30 min at 4°C. Protein concentrations were measured by the Bradford method and 50 µg samples were mixed with 2x Laemmli buffer, boiled for 5 min, electrophoresed in 14% Tris–Glycine gels and then were transferred to Immobilin membranes (Millipore, Bedford, MA). Western blot analyses were performed as described previously (41) using polyclonal antibodies to rhoA and rhoB (Santa Cruz Biotechnologies, Santa Cruz, CA) at a concentration of 1:100 v/v. Detection of antibody binding was done using I125-labeled protein A and subsequent imaging was performed on Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis
The data from western blots were analyzed using control values as 100% and then calculating the changes in the amount of protein. Subsequently the data from different blots (expressed as percentage of control samples) were pooled for statistical analysis using Student's t-test, P < 0.05 was taken as significant. Data are presented as mean ± standard deviation.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
References
 
Lovastatin causes morphologic changes and apoptosis in IEC-18 cells
Fifty percent confluent cultures of parental IEC-18 cells, vector control C1 or k-ras transformed clones R1, R4, R8 and R10 were incubated with lovastatin at a concentration of 10–30 µM. After 12 h, cell rounding and detachment were observed. Eighteen hours after addition of lovastatin 30 µM, ~80% of the cells had detached from the culture plates. IEC-18 parental cells and k-ras transformed clones R1 and R10 underwent similar rounding and detachment after lovastatin treatment (Figure 1AGo).




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  		Fig. 1. Lovastatin induces cytoskeletal changes and apoptosis in IEC-18 cells. (A) Photomicrographs of IEC-18, R1 and R10 cells after lovastatin treatment. Exponentially growing cells were incubated with lovastatin 10 µM. Photographs of the unstained cells growing in culture plates were taken at 18 h after lovastatin was added with a Nikon Invertoscope at 400x magnification. (i) untreated parental IEC-18 cells; (ii) IEC-18 cells exposed to lovastatin 10 µM; (iii) untreated IEC-18-R1 cells; (iv) IEC-18-R1 cells treated with lovastatin 10 µM; (v) Untreated IEC-18-R10 cells; (vi) IEC-18-R10 cells treated with lovastatin 10 µM. Note: the lovastatin treated cells become rounded in shape, detach from the surface of the culture plates and float as singlets. (B) Comparison of apoptosis induced by lovastatin in IEC-18 parental cells and k-ras transfected clones. Parental IEC-18 (P), vector control C1, R1, R4, R8 and R10 cells were incubated with 30 µM lovastatin and cells collected for quantification of apoptosis by flow cytometry at 24 h. Experiments were repeated three times and similar results were consistently observed. (i) Apoptosis quantified as % sub-diploid cells. (ii) Apoptosis quantified as % TUNEL positive cells.

 
After 24 h of incubation with lovastatin, cells were harvested for flow cytometry. Apoptosis was quantified by measuring the fraction of sub-diploid cells or measuring the fraction of TUNEL positive cells (Figure 1BGo). Lovastatin induced apoptosis in the parental IEC-18, vector control C1, ras transformed R1, R4 and R8 cells. In contrast, the IEC-18-R10 clone, which lacks functional p53 protein, was very resistant to lovastatin-induced apoptosis.

Effect of isoprenoids on lovastatin-induced apoptosis
By inhibiting HMG-CoA reductase, lovastatin prevents conversion of HMG-CoA to mevalonate and thereby inhibits the formation of isoprenoids (farnesyl pyrophosphate and geranylgeranyl pyrophosphate). To study the relative importance of farnesylation and geranylgeranylation in lovastatin-induced cell rounding, detachment and apoptosis, add-back experiments were done using mevalonate, farnesylpyrophosphate or geranylgeranylpyrophosphate along with lovastatin. Mevalonate 100 µM or geranylgeranylpyrophosphate 1–10 µM prevented the cell rounding, detachment and apoptosis induced by lovastatin in IEC-18 (Figure 2Go) and R10 cells, while farnesylpyrophosphate 100 µM was only partially effective. Thus, inhibition of geranylgeranylation is the probable mechanism of action of lovastatin.




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  		Fig. 2. Effect of addition of isoprenoids upon lovastatin-induced morphological changes and apoptosis. IEC-18 cells were incubated with lovastatin 30 µM ± mevalonate 100 µM, farnesyl pyrophosphate 100 µM or geranylgeranyl pyrophosphate 10 µM. After 24 h, photomicrographs of the cells were taken. Cells were then harvested for flow cytometry to quantitate apoptosis. Apoptosis was quantified as percent sub-diploid cells. Experiments were repeated three times and similar results were consistently observed. (A) Photomicrographs of the parental IEC-18 cells (photomicrographs i–v). (i) Untreated parental IEC-18 cells; (ii) +lovastatin; (iii) +lovastatin and mevalonate; (iv) +lovastatin and FPP; (v) +lovastatin and GGPP. (B) Effect of addition of isoprenoids upon lovastatin-induced apoptosis.

 
Lovastatin-induced morphologic changes precede apoptosis and can be reversed by addition of geranylgeranylpyrophosphate
To determine if the morphologic changes induced by lovastatin are secondary to apoptosis, we determined the temporal relationship between them. GGPP was added to lovastatin treated cells at t = 0 and t = 12 h and cells collected for flow cytometry at 24 h. We found that the morphologic changes appeared at 12 h, at which time only minimal increase in apoptosis was observed on flow cytometry (Figure 3A and BGo). To rule out the possibility that, at 1 h, lovastatin treated cells were in very early phase of apoptosis and therefore not counted as apoptotic by flow cytometry, we added geranylgeranylpyrophosphate to lovastatin treated cells after appearance of morphologic changes (i.e. 12 h). Geranylgeranylpyrophosphate caused reattachment of lovastatin-treated cells to the culture plates with resumption of normal morphology, prevented apoptosis measured at 24 h and caused the cells to proliferate again. These data indicate that the morphologic changes induced by lovastatin are reversible and are not secondary to apoptosis.



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  		Fig. 3. Geranylgeranylpyrophosphate reverses lovastatin-induced morphologic changes, proliferation arrest and prevents apoptosis in IEC-18 cells. IEC-18 cells were incubated with lovastatin 30 µM and GGPP 10 µM was added at t = 0 or t = 12 h. Floating and adherent cells were harvested at 24 and 48 h for measurement of cell counts and for flow cytometry to quantitate apoptosis. (A) Photomicrographs (400x). (i) Untreated cells at t = 0 h; (ii) lovastatin treated cells after 12 h incubation; (iii) lovastatin treated cells after 24 h incubation, to which GGPP was added after 12 h. (B) Effect of addition of GGPP on lovastatin-induced apoptosis. (C) Effect of GGPP on proliferation (cell counts) in lovastatin treated cells (mean ± SD). Note: lovastatin induces morphologic changes and cell detachment at 12 h. GGPP added at 12 h causes cells to reattach to culture plates and resume normal morphology, prevents apoptosis and restores proliferation.

 
Clostridium difficile toxin B induces morphologic changes and apoptosis similar to lovastatin in IEC-18 cells
To determine whether inactivation of rho by alternate mechanisms could induce similar cytoskeletal changes and apoptosis, we compared the effects of C. difficile toxin B (which directly inactivates rho by glucosylation) and lovastatin (which inactivates rho by inhibiting isoprenylation) in IEC-18 cells and its k-ras transformed clones. C. difficile toxin B causes cell rounding and detachment and induces apoptosis in IEC-18 cells very similar to lovastatin (Figure 4Go). Adherent cells, after treatment with lovastatin or C. difficile toxin B, showed remarkably similar morphology including cellular retraction (Figure 5AGo). Cell rounding and detachment started between 2–4 h after incubation at higher C. difficile toxin B concentrations (>10 pM) and between 4–8 h at lower concentrations (1–10 pM). R10 cells also were relatively resistant to apoptosis induced by C. difficile toxin B (Figure 4Go).



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  		Fig. 4. Effect of Clostridium difficile toxin B upon IEC-18 cells. IEC-18 parental and R10 cells were treated with C. difficile toxin B 1, 3 or 10 picomolar. Cells were harvested after 24 h incubation for quantification of apoptosis by flow cytometry. Apoptosis was quantified as percent sub-diploid cells. Note: C. difficile toxin B causes dose-dependent apoptosis in IEC-18 cells but R10 cells were resistant to apoptosis. Experiments were repeated three times and similar results were consistently observed.

 



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  		Fig. 5. Effect of cycloheximide upon lovastatin-induced changes in IEC-18 cells. IEC-18 cells were treated with lovastatin 30 µM or C. difficile toxin B 10 pM ± cycloheximide 10 µg/ml for 24 h. Cells were then photographed and harvested for flow cytometry for quantification of apoptosis as percent sub-diploid cells. (A) Photomicrographs of the IEC-18 cells at 1000x magnification. (i) Untreated IEC-18 cells; (ii) +cycloheximide; (iii) +lovastatin; (iv) +lovastatin and cycloheximide; (v) +C. difficile toxin B; (vi) +C. difficile toxin B and cycloheximide. (B) Effect of cycloheximide on lovastatin-induced apoptosis. (C) Effect of cycloheximide on C. difficile toxin B-induced apoptosis. All experiments were repeated three times and similar results were consistently observed.

 
Cycloheximide inhibits the cellular effects of lovastatin but not C. difficile toxin B in IEC-18 cells
The protein synthesis inhibitor, cycloheximide (10 µg/ml) when added with lovastatin 30 µM completely prevented cell rounding and detachment in parental IEC-18 cells and also in R10 cells (Figure 5AGo). In IEC-18 parental cells, cycloheximide also inhibited lovastatin-induced apoptosis (Figure 5BGo). In contrast, cycloheximide 10 µg/ml, added along with Clostridium difficile toxin B 1, 3 or 10 pM to IEC-18 cells did not prevent morphologic changes (Figure 5AGo) or apoptosis (Figure 5CGo).

Cycloheximide inhibits lovastatin-induced increases in cytosolic rhoB but does not restore changes in its membrane association
To determine if restoration of membrane translocation of rho was necessary and sufficient to inhibit lovastatin-induced changes that were observed, we tested if geranylgeranylpyrophosphate or cycloheximide prevent lovastatin-induced changes in membrane-associated rho. IEC-18 cells were treated with lovastatin 30 µM ± geranylgeranylpyrophosphate 10 µM or cycloheximide 10 µg/ml for 12 h. Cells were harvested and then separated into membrane and cytosolic fraction. Lovastatin 30 µM reduced the amount of both membrane-bound rhoA and rhoB, which were partially restored by addition of geranylgeranyl pyrophosphate 10 µM but not by cycloheximide 10 µg/ml (Figure 6AGo).




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  		Fig. 6. Effect of lovastatin on cytosolic and membrane-bound rhoA and rhoB. IEC-18 cells were treated with lovastatin 30 µM ± geranylgeranyl pyrophosphate 10 µM or cycloheximide 10 µg/ml. Cells were harvested after 12 h incubation and separated into membrane fraction and cytosolic fraction. Western blotting was done to quantify changes in amount of rhoA and rhoB bound to membrane and in the cytosol. (A) Changes in membrane-bound rhoA and rhoB after lovastatin treatment (mean ± SD). (B) Changes in cytosolic rhoA and rhoB after lovastatin treatment (mean ± SD). P < 0.05 was taken as significant.

 
Lovastatin 30 µM caused a threefold increase in cytosolic rhoB which was prevented by geranylgeranyl pyrophosphate or cycloheximide (Figure 6BGo). There was no change in the amount of cytosolic rhoA after adding lovastatin. However, cycloheximide reduced cytosolic rhoA in lovastatin treated cells (P = 0.02 versus controls and P = 0.01 versus lovastatin only).

Discussion

The present studies demonstrate that lovastatin, an HMG-CoA reductase inhibitor, induces cell rounding, detachment and apoptosis in IEC-18 cells, a spontaneously immortalized rat intestinal epithelial cell line. These morphologic changes and apoptosis appeared to result from inhibition of geranylation of intracellular proteins. The morphologic changes preceded apoptosis and were reversed by addition of geranylgeranylpyrophosphate. The degree of lovastatin-induced apoptosis seems unrelated to the presence of k-ras mutations. Clostridium difficile toxin B, which directly inactivates rho by glucosylation, induced very similar morphologic changes and apoptosis in the IEC-18 cells. However, the R10 clone, that lacks functional p53, was relatively resistant to lovastatin or C. difficile toxin B-induced apoptosis. Cycloheximide prevented the cell rounding, detachment and apoptosis induced by lovastatin but not that induced by C. difficile toxin B. Lovastatin reduced membrane-bound rhoA and rhoB, which were partially restored by geranylgeranylpyrophosphate but not by cycloheximide. Lovastatin-treated cells also showed a three-fold increase of rhoB in the cytosol which was prevented by addition of either geranylgeranylpyrophosphate or cycloheximide.

The role of ras mutations in determining the sensitivity of various cell types to lovastatin has been debated (42). Early studies attributed inhibition of ras farnesylation as the predominant mechanism of action of lovastatin and some studies suggested that H-ras transformation increases the sensitivity to lovastatin (43). In our studies, parental IEC-18 cells and their k-ras transfected clones showed similar cytoskeletal changes and apoptosis with lovastatin. Thus, lovastatin could potentially be an adjunct to chemotherapy even in tumors which do not have mutated overactive ras. IEC-18-R10 cells, which lack functional p53 protein, were however relatively resistant to apoptosis induced by lovastatin or C. difficile toxin B, suggesting that apoptosis induced by these agents is p53-dependent or p53 sensitive. This is consistent with an earlier report that lovastatin induced neuronal apoptosis is accompanied by p53 as well as bax induction (44).

Both morphologic changes (cell rounding and cellular detachment) and apoptosis induced by lovastatin were completely inhibited by geranylgeranyl pyrophosphate 10 mM, but not by farnesyl pyrophosphate. Thus, inhibition of geranylgeranylation appears to be the predominant mechanism of lovastatin-induced apoptosis. Earlier studies in prostate stromal cells and human promyelocytic cells have also found that geranylgeranyl pyrophosphate prevents lovastatin-induced apoptosis (4,5). Since ras is primarily farnesylated, these observations also imply that inhibition of ras-isoprenylation is not the predominant mechanism for lovastatin-induced cell death.

Cell rounding and detachment due to lovastatin or C. difficile toxin B are not secondary to apoptosis, since IEC-18-R10 cells undergo similar morphologic changes without undergoing apoptosis and in parental IEC-18 cells these morphologic changes preceded apoptosis. Geranylgeranylpyrophosphate added after the appearance of cell rounding and detachment induced cells to reattach to the culture plates, restored their morphology and prevented apoptosis, confirming that the morphologic changes are reversible and not due to apoptosis.

The proteins of the rho family are involved in diverse biological functions including cell growth, transformation, cell motility and migration, metastasis and response to stress. RhoA is overexpressed in several tumors (45) and the oncogenes vav and ost function as guanine exchange factors for rho proteins (46,47). The crucial role of rho family members in determining cytoskeletal changes and apoptosis induced by lovastatin is suggested by the following observations: (i) geranylgeranyl-PP but not farnesyl-PP prevented lovastatin-induced cytoskeletal changes and apoptosis; (ii) Clostridium difficile toxin B, which directly inactivates rho, rac and cdc42 by glucosylation, also induced cell detachment, morphologic changes and apoptosis in IEC-18 cells, identical to that induced by lovastatin; (iii) C3-exotoxin which inactivates rho also induces cell rounding, detachment and apoptosis (30); (iv) lovastatin-induces morphologic changes in a different cell type, NIH3T3 cells which are reversed by the microinjection of a dominant active rhoA. Thus, as discussed above, lovastatin-induced morphologic changes and apoptosis appear to be two discrete phenomena. If microinjection of Val14RhoA restores morphologic changes and also prevents apoptosis (48), inactivation of rhoA seems critical for both these phenomena.

As inactivation of rhoA is critical for lovastatin-induced morphologic changes and apoptosis and membrane translocation of rho is crucial for its normal function, then the following questions arise: (i) if inhibition of membrane translocation of rhoA by lovastatin is necessary and sufficient to cause the morphologic changes and apoptosis; (ii) if restoration of membrane bound rho was necessary to prevent these changes. In our studies, geranylgeranyl pyrophosphate partially restored the amount of membrane bound rhoA and rhoB confirming the data from other studies (49) that rho isoprenylation plays an important role in its membrane translocation. However cytoskeletal changes and apoptosis were completely prevented by GGPP even when rho translocation was only partially restored. Furthermore, cycloheximide failed to restore the amount of membrane-bound rhoA or rhoB but completely prevented lovastatin induced morphologic changes and apoptosis. These data imply that lovastatin-induced morphologic changes and apoptosis are unrelated to inhibition of membrane translocation of rhoA or rhoB. Similarly, in lovastatin-treated NIH3T3 cells Koch et al. (50) found that microinjection of Val14rhoA caused a reversion to a normal morphology, although the amount of membrane bound rho was not restored.

Membrane bound rhoA and rhoB decreased in cells treated with lovastatin and cycloheximide, suggesting that even in the presence of cycloheximide, lovastatin inhibits rho isoprenylation and its membrane translocation. As cycloheximide prevents apoptosis without restoring isoprenylation, this implies that apoptosis in lovastatin-treated cells is not due to depletion of isoprenylated rhoA but rather due to synthesis or accumulation of a protein that inactivates rhoA. We speculate that cycloheximide blocks synthesis of this protein(s) that is required for lovastatin effect. Cycloheximide does not inhibit morphologic changes and apoptosis induced by Clostridium difficile toxin B as unlike lovastatin, this toxin directly inactivates rhoA by a separate mechanism involving glucosylation of rho (Figure 7Go). Lovastatin treated cells showed a three-fold increase in rhoB without significant changes in the amount of rhoA, which was prevented either by geranylgeranylpyrophosphate or by cycloheximide. Imbalance between rhoA, rac1 and cdc42 signaling has previously been shown to induce changes in cytoskeletal morphology (51). A very recent preliminary report suggests that the farnesyl transferase inhibitors, which also inhibit geranylgeranylation of rho inhibit tumor growth by upregulating transcription and expression of rhoB (52). It is conceivable that an imbalance between relative amounts of rhoA and rhoB results in apoptosis. The precise mechanism by which inhibition of geranylgeranylation of rho by lovastatin induces accumulation of rhoB and whether rhoB has a dominant negative effect on rhoA and potential mechanism(s) of this effect need to be determined and are under investigation in our laboratory.



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  		Fig. 7. Proposed mechanism to explain the inhibitory effects of cycloheximide on lovastatin-induced morphologic changes and apoptosis.

 
In summary, lovastatin causes cell rounding, detachment and apoptosis in IEC-18 cells by inhibiting geranylgeranylation of rho, and k-ras transformation does not alter the sensitivity of these cells to lovastatin. Morphologic changes induced by lovastatin are reversible and not secondary to apoptosis. Lovastatin-induced cellular changes are not related to inhibition of membrane-translocation of rho. We speculate that lovastatin induced cell rounding, detachment and apoptosis may be related to accumulation of yet unidentified protein(s) that have a dominant negative effect on rho function.


   Notes
 
* Both authors contributed equally to this publication. Back

5 Division of Gastroenterology, St Luke's-Roosevelt Hospital Center, 1111 Amsterdam Ave, New York, NY 10025, USA Email: pholt{at}slrhc.org Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 

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Received July 10, 2001; revised December 12, 2001; accepted December 19, 2001.


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