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Clinical Cancer Research Vol. 5, 2596-2604, September 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

RB Status as a Determinant of Response to UCN-01 in Non-Small Cell Lung Carcinoma1

Philip C. Mack, David R. Gandara, Cai Bowen, Martin J. Edelman2, Teresa Paglieroni, Joachim B. Schnier, Edward P. Gelmann and Paul H. Gumerlock3

Cancer and Molecular Research Laboratory, Division of Hematology/Oncology, Departments of Internal Medicine [P. C. M., D. R. G., P. H. G.] and Medical Biochemistry [J. B. S.], University of California, Davis, Sacramento, California 95817; Northern California Veterans Administration Systems of Clinics, Martinez, California 94553 [D. R. G., M. J. E.]; the Sacramento Medical Foundation Center for Blood Research, Sacramento, California 95816 [T. P.]; and Division of Medical Oncology, Lombardi Cancer Research Center, Georgetown University School of Medicine, Washington DC 20007 [C. B., E. P. G.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calu1), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The elucidation of the molecular events involved in the regulation of cell cycle progression has led to identification of a complex web of interacting genes and their protein products. Prominent within this web are the tumor suppressor genes RB and p53 (also known as TP53; Refs. 1, 2, 3 ). In cycling cells, activated complexes of G1 phase cyclins and Cdks phosphorylate Rb,4 resulting in the release of transcription factors necessary for the onset of and progression through S-phase (2, 3, 4, 5, 6) . In contrast, Cdk inhibitors, such as p16CDKN2 (Mts1) and p21CDKN1 (Waf1/Cip1), interfere with cyclin/Cdk activity, leading to cell cycle arrest at the G1 checkpoint (7, 8, 9, 10) . For example, the p53 pathway is activated in response to DNA damage, resulting in p53-dependent induction of p21CDKN1 and G1 arrest (9 , 10) . Mutations in cell cycle genes are common to most cancer types, including NSCLCs, suggesting potentially exploitable differences in checkpoint arrest between normal and malignant cells (11, 12, 13) .

Novel antineoplastic agents that modulate cell cycling are in development. One such agent is the protein kinase inhibitor, UCN-01. UCN-01 exhibits potent in vitro and in vivo anticancer activity against a broad range of murine tumors, human cancer cell lines, and xenografts and is in early Phase I clinical trials (14, 15, 16, 17) . UCN-01 has also been shown to potentiate the anticancer activity of cisplatin and other DNA-damaging agents (16, 17, 18, 19) .

UCN-01 was originally characterized as an inhibitor of PKC (20) . However, its anticancer activity is more likely to result from modulation of cell cycle progression than the direct effect of PKC inhibition. Experiments comparing the effects of UCN-01 and GF 109203X, a selective PKC inhibitor, demonstrated that although both compounds inhibited PKC activity, UCN-01 had a disproportionately greater effect on cell growth inhibition (21) . Initial studies have indicated that UCN-01 treatment results in inhibition of Cdk2, Cdk4, and Cdk6, hypophosphorylation of Rb, and the accumulation of cells in G1 (15 , 22, 23, 24) . Loss of Cdk activity may result from either direct inhibition by UCN-01 or induction of the Cdk inhibitors p21CDKN1 and p27KIP1 (24) .

Molecular alterations that contribute to tumorigenesis by disrupting normal cell cycle regulation may also confer selective resistance to classic chemotherapeutic agents, many of which modulate the cell cycle. We hypothesized that the anticancer activity of UCN-01 is dependent on the molecular status of key cell cycle regulatory genes and the ability to induce Cdk inhibitors, Rb hypophosphorylation, and G1 arrest. To address this, we investigated the effects of UCN-01 on growth and modulation of gene expression in human NSCLC cell lines with differential abnormalities of p16CDKN2, RB, and p53.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures.
The NSCLC cell lines A549, Calu1, and H596 were acquired from the American Type Culture Collection (Rockville, MD). The normal human fetal lung fibroblast strain T3891 was initiated in our laboratory (25) . Human isogenic models of RB expression, the bladder cancer cell line 5637 (RB-null), and the prostate cancer cell line DU145 (RB mutant) have been reported previously (26 , 27) . Cells were cultured in either RPMI 1640 or DMEM (BioWhittaker, Walkersville, MD) with 10% heat-inactivated fetal bovine serum, supplemented with penicillin and streptomycin. Cells were maintained at 37°C in 5% CO2 in air.

Drug Treatment.
Stock solutions were prepared by dissolving UCN-01 in DMSO at a concentration of 1 mg/ml and were stored at -20°C. Immediately prior to treatment, the UCN-01 stock solution was further diluted in serum-free media. UCN-01 was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute.

The protein extracts for the Western blots and the cells for flow cytometry from untreated control cultures were both obtained from log phase cells at ~60% confluency. Similarly, the cultures exposed to drugs were treated at ~40%–60% confluency and harvested at the respective time points.

Western Blotting.
Soluble protein extracts were prepared from cell pellets in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% NP40, 1 mM phenylmethylsulfonyl fluoride, 5 mM NaF, and 1 mM sodium o-vanadate. DNase (20 units/ml) was added, and the mixture was placed on ice for 30 min with occasional vortexing. The lysate was cleared by centrifugation at 12,000 rpm in a microfuge for 15 min at 4°C. Protein concentrations were quantitated from duplicate readings using a modified Bradford assay (Bio-Rad Laboratories, Richmond, CA) on a 96-well plate reader. Protein samples were diluted with lysis buffer to either 20 or 30 µg/µl to facilitate equal loading of samples. SDS-PAGE gels were cast in a discontinuous fashion using a 4% stacking gel with slight modification of the methods of Laemmli, as reported previously (25 , 28) . Rb was analyzed on a 7.5% resolving gel (Hoefer Scientific Instruments, San Francisco, CA). p16CDKN2 and p21CDKN1 were separated on a 15% gel using a mini-gel system (Protean II; Bio-Rad Laboratories). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) at a constant 125V for 70 min.

Membranes were blocked in a solution consisting of 5% blocking grade nonfat dry milk (Bio-Rad Laboratories) in TBST [25 mM Tris-HCl (pH 8.0), 31.25 mM NaCl, and 0.025% Tween 20]. Blots were incubated either overnight at 4°C or at room temperature for 1 h with one of the following primary antibodies: anti-Rb mouse monoclonal (G3-245), anti-p16CDKN2 rabbit polyclonal, or anti-p21CDKN1 mouse monoclonal (6B6; all from PharMingen, San Diego, CA). Blots were washed three times in the blocking solution (15 min each) and incubated in a 1:1250 dilution of the appropriate biotinylated (p16CDKN2 and p21CDKN1) or horseradish peroxidase-conjugated (Rb) secondary antibody (Vector Laboratories, Burlingame, CA or Promega Corp., Madison, WI) for 1 h. Blots were washed once in blocking solution and three times in TBST (10 min each). Blots using biotinylated secondary antibodies were incubated with streptavidin-horseradish peroxidase (Boehringer Mannheim, Indianapolis, IN) diluted to 1:1000 in TBST for 30 min, followed by two washes in TBST. All blots were washed in TBS (TBST lacking Tween) for 5 min and incubated with chemiluminescent detection reagents (ECL; Amersham, Arlington Heights, IL) as per instructions of the manufacturer. Finally, membranes were exposed to Kodak XAR films. After development, blots were stained with Ponceau S total protein stain (Sigma Chemical Co., St. Louis, MO) to confirm equivalent transfer of proteins. The protein bands of interest were compared with expression of the endogenous control ß-actin detected with antibody clone AC15 (Sigma).

Growth Curves.
Cell growth was assessed using the MTT assay (Sigma Chemical Co.; Ref. 29 ). Each experiment consisted of five or six replicate wells at each treatment dose, and each experiment was performed at least four times. Cells were plated on 96-well plates at a density of 5000 cells/well. One day after plating, cells were treated with UCN-01 at concentrations ranging from 10 to 1000 nM. After drug exposure for 24 h, the UCN-01 containing medium was removed, and the wells were washed and replaced with fresh medium. When control cells reached 90% confluence, 25 µl of MTT (2 mg/ml in PBS, pH 7.4) was added to each well and incubated for 3 h at 37°C. The medium was then removed, and 175 µl of DMSO were added and incubated for 15 min while agitated on an orbital rotator. Signal from the MTT dye was read by absorbance using the E-max microplate reader with Softmax software (Molecular Devices, Sunnyvale, CA). Absorbance readings were converted to cell numbers by comparison with the standard curves (four-parameter fittings) for each cell line and then divided by the cell number in the untreated control wells to obtain the fraction of cells present after treatment with each dose of UCN-01. The fraction affected was equal to 1.0 minus the fraction present in the treated wells. These data were then analyzed using Calcusyn software (Biosoft, Cambridge, United Kingdom) to determine the concentration at which cell growth is inhibited by 50% (IC50).

Flow Cytometry.
Cells were pelleted and resuspended in fixative (95% methanol, 5% glacial acetic acid) for 3 or more days. One day prior to analysis, cells were washed in PI stain, pelleted, and resuspended in stain according to the method of Deitch et al. (30) . PI-stained samples were filtered through 50 µm nylon mesh to remove aggregates before analysis on a flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems Inc., San Jose, CA) equipped with a doublet discrimination module, a 15 mW argon laser tuned to 488 nm, and acquisition and analysis software (CellFIT; Becton Dickinson). The electronics of the instrument were adjusted using PI-stained chicken erythrocyte nuclei so that the modal channel for the diploid G0–G1 nuclei would be in channel 300 (on a 0–1023 arbitrary unit linear scale). The analysis of each cell line was optimized to similar conditions. Machine performance and alignment were verified using unstained 2 µm beads, PI-stained chicken erythrocyte nuclei, and PI-stained calf thymus nuclei. Twenty thousand events were collected for each analysis. Cell doublets were excluded by gating on a histogram of FL-2 width versus FL-2 area. Analysis of single-parameter DNA histograms for the distribution of cells in G0–G1, S, and G2-M cell cycle phases was performed on gated events using the RFIT mathematical calculation model of the software program.

For 5637 and RB5, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, followed by 70% ethanol, and stored at -20°C. Cells were washed twice with 0.1% BSA in HBSS and then washed with 0.1% Triton X-100-HBSS and resuspended in 1.0 ml of PI-RNaseA (PI, 5 µg/ml; RNaseA, 50 units/ml in HBSS). Flow cytometry for DU-145 and the B5 and DU1.1 sublines was conducted using a protocol described previously (27) .

Statistics.
Statistical comparison of growth inhibition data were conducted using Bonferroni’s Multiple Comparison Test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
UCN-01 Inhibits Growth of NSCLC Cell Lines with Wild-Type RB.
To correlate the growth response after UCN-01 treatment with the molecular status of selected cell cycle regulatory genes, human NSCLC cell lines with differential abnormalities of p16CDKN2, p53 and RB were evaluated in comparison to the normal human fetal lung fibroblast strain, T3891 (Table 1)Citation . A549 cells are p16CDKN2-null, Calu1 cells are p53-null, and H596 cells are RB-null and additionally harbor a p53 point mutation (codon 245, GGC->TGC, Gly->Cys, and loss of heterozygosity; Refs. 12 , 13 , 31, 32, 33 ). We have shown previously that in Calu1 cells, p16CDKN2 protein is undetectable, despite the presence of mRNA by reverse transcription-PCR (34) . Further characterization by Western blotting of A549 and H596 cells verified the absence of protein expression of p16CDKN2 and Rb, respectively (data not shown).


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  		Table 1 NSCLC mutational status and UCN-01 IC50

 
To examine the involvement of p16CDKN2 in the response to UCN-01, A549 and T3891 cells were compared. Treatment resulted in a dose-dependent inhibition of cell growth with no significant difference between these p16CDKN2-null and the normal cells (P > 0.05; Fig. 1Citation ). Thus, in this model, p16CDKN2 is not required for UCN-01-induced growth inhibition. Because of the established roles of p53 in cell cycle regulation and p21CDKN1 induction, we examined the consequence of mutant p53 in the response to UCN-01 by comparing Calu1 with A549 and T3891. Calu1 cells (p53-null) were also responsive to UCN-01 treatment (Fig. 1)Citation . Although Calu1 cells were somewhat less inhibited, there were no significant differences when the responses of T3891 or A549 versus Calu1 were compared (P > 0.05 in both cases). Thus, p53 is not required for UCN-01-induced growth inhibition. In contrast, H596 cells (RB-null) were more refractory to UCN-01 treatment (Fig. 1)Citation . Using these data, the extrapolated IC50 of H596 was 3025 nM. The difference in growth response between this cell line and each of the others was statistically significant (P < 0.001 in all cases), suggesting the possibility that RB status influences growth response to UCN-01. IC50s from these experiments are correlated with the molecular status of p16CDKN2, p53, and RB of these cell lines in Table 1Citation .



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  		Fig. 1. UCN-01-induced growth inhibition of NSCLC. Effect of UCN-01 on T3891, A549, H596, and Calu1 cells after 24-h treatments with UCN-01 (concentration indicated on X-axis versus Fraction Affected on Y-axis) in relation to untreated controls as measured by the MTT assay as described in "Materials and Methods." T3891, A549, and Calu1 cells show dose-dependent inhibition. Growth of H596 cells are only modestly inhibited by UCN-01, even at concentrations up to 1000 nM. All points represent a minimum of four separate experiments with five determinations/point/experiment. Bars, ±SD.

 
UCN-01 Treatment Results in Rb Hypophosphorylation.
To investigate changes in the phosphorylation state of Rb, T3891, and A549 cells were cultured with 100, 300, and 600 nM concentrations of UCN-01 for both 24 and 72 h. These treatments resulted in a shift of Rb to the hypophosphorylated state (Fig. 2A)Citation . The Rb in T3891 cells was completely hypophosphorylated by 100 nM UCN-01 at 24 h. In A549 cells, Rb hypophosphorylation was both time and dose dependent, showing complete hypophosphorylation at 72 h of 600 nM UCN-01. Changes in Rb phosphorylation in Calu1 cells were investigated at a dose of 500 nM UCN-01 for 24 h. Hypophosphorylation was observed at this dose (Fig. 2B)Citation . To determine the time dependence of Rb hypophosphorylation, T3891 cells were treated with 100 nM of UCN-01 for 12 h. Hypophosphorylation of Rb was nearly complete at this time point (Fig. 2C)Citation .



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  		Fig. 2. UCN-01 treatment results in Rb hypophosphorylation. A, Western blot of Rb protein from control T3891 and A549 cells and cells cultured with UCN-01 at 100, 300, and 600 nM concentrations for 24 and 72 h. Rb in T3891 cells is completely hypophosphorylated after 24 h of 100 nM UCN-01 treatment and remains so at longer exposures and at higher concentrations. A549 cells show a partial shift in Rb phosphorylation that is both dose and time dependent and eventually reaches complete hypophosphorylation at 72 h of 600 nM UCN-01. B, Rb is hypophosphorylated in Calu1 cells treated with 500 nM of UCN-01 for 24 h. C, T3891 cells show complete Rb hypophosphorylation after a 12-h exposure to UCN-01.

 
p16CDKN2 Protein Expression Is Not Induced by UCN-01 Treatment.
Induction of p16CDKN2 expression after treatment with UCN-01 was examined by Western blotting. In T3891 cells, p16CDKN2 showed no substantial change after 24 h of 100 nM UCN-01 (Fig. 3)Citation .



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  		Fig. 3. UCN-01 has no effect on p16CDKN2 expression. Western blot of T3891 cells after UCN-01 treatment at 100 nM, 24 h (Lane M,Mr 21,000 protein standard).

 
UCN-01 Induction of p21CDKN1 Is p53 Independent.
Protein expression of the CDK inhibitor p21CDKN1 was assessed after 24 h treatment with UCN-01 at 100 and 500 nM concentrations. Induction of p21CDKN1 was evident in all cell lines examined (Fig. 4)Citation . In A549 and H596 cells, basal expression of p21CDKN1 was observed, which was further induced after treatment. In the p53-null Calu1 line, basal expression was not observed; however, after UCN-01 treatment, p21CDKN1 was readily detectable, indicating that UCN-01-induced expression of p21CDKN1 is p53 independent. Induction of p21CDKN1 was also observed in the normal fibroblast strain T3891 (data not shown).



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  		Fig. 4. UCN-01 induces p21CDKN1 expression in both wild-type p53 and mutant p53 NSCLC cells. Composite Western blot of p21CDKN1 from A549, Calu1, and H596 cells after UCN-01 treatment at 100 and 500 nM for 24 h. Protein expression of p21CDKN1 is induced in NSCLC lines that are wild-type p53 (A549), p53-null (Calu1), and p53 mutant (H596). ß-actin expression (lower panel) was used as an endogenous reference gene standard.

 
NSCLC Cells with Wild-Type RB Arrest in G1 after UCN-01 Treatment, Independent of p53 Status.
The DNA content of individual cells was analyzed to examine cell cycle modulation after exposure to UCN-01. After UCN-01 treatment, T3891 and A549 cells showed a near complete absence of cells in S-phase and an accumulation of cells in G0–G1 (Fig. 5, A and B)Citation . T3891 cells treated with UCN-01 showed a percentage of cells in G2 (10% or less) as did A549 cells, whereas cells arrested by serum starvation accumulated almost exclusively in the G0–G1 phase. UCN-01-treated Calu1 cells (p53-null) showed a decrease of ~30% of cells in S-phase and an equivalent increase of cells in the G0–G1 phase and a small G2 peak (data not shown). The RB-null NSCLC line H596 was also investigated for changes in cell cycle response. At both the 100 and 500 nM dose, the percentage of cells in S-phase was largely unchanged, indicating continuing exit from G1. In addition, a substantial decrease in cells in G2-M was noted (Fig. 5C)Citation .



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  		Fig. 5. UCN-01 treatment results in G1 checkpoint arrest. Flow cytometric analysis was used to measure DNA content in control and UCN-01-treated cells as described in "Materials and Methods." A, T3891 cells treated with 100 nM of UCN-01 for 24 h. Serum starvation was used as a reference standard for cell cycle arrest. B, A549 cells treated with 600 nM UCN-01 for 48 h. Listed below the histograms are the percentages of cells in each phase of the cell cycle. C,RB-null H596 cells treated with 100 and 500 nM of UCN-01 (24 h) showed loss of G2 phase cells, while maintaining S-phase cell levels.

 
RB Status Influences UCN-01-induced G1 Arrest.
To further test the role of RB in UCN-01 response, we investigated the effects using two established human isogenic models of Rb expression, the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). These cell lines were each stably transfected with wild-type Rb, yielding the 5637 subline RB5 and the DU-145 subline DU1.1 and B5 (26 , 27) . The expressed Rb protein in RB5 was hypophosphorylated after treatment with both 100 and 500 nM UCN-01 (Fig. 6A)Citation . At 100 nM UCN-01, RB5 cells showed marked accumulation in G1 phase in contrast to the RB-null parent line 5637, demonstrating the role of Rb in G1 arrest in response to UCN-01 (Fig. 6B)Citation . G1 accumulation in the parent line became apparent only at 500 nM.



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  		Fig. 6. A, UCN-01-induced Rb hypophosphorylation in RB-transfected 5637 cells. The RB-expressing RB5 subline was treated with UCN-01 at 100 or 500 nM for 48 h. Treatment resulted in Rb hypophosphorylation at both doses. B, UCN-01-induced G1 arrest in RB5 cells. Treatment with UCN-01 at 100 or 500 nM for 24 h resulted in accumulation of cells in the G1 phase of the cell cycle in the RB-expressing 5637 subline B5. In parent 5637 cells, modest G1 arrest is seen when treated with 500 nM UCN-01.

 
The prostate carcinoma model of Rb expression revealed similar results at 500 nM of UCN-01 for 30 h (Table 2Citation ; Fig. 7Citation ). Increases in G1 accumulation were greater in the sublines (DU1.1, +32%; B5, +27%; average, 29.5%) than in the parent (DU-145, +20%). Similar results were obtained from cells treated with 100 nM UCN-01 for 72 h. Decreases in percentages of cells in S-phase from the two sublines expressing wild-type Rb (DU1.1, -24%; B5, -18%; average, -21%) were also greater than the RB-mutant parent line (DU-145, -14%). These data further support a role for wild-type RB in G1 arrest in response to UCN-01.


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  		Table 2 Cell cycle analysis of DU-145 and wild-type (wt) RB-transfected sublines DU1.1 and B5 treated with UCN-01

The table indicates the percentage of cells in cell cycle compartments (G0–G1, S, and G2-M). At 100 nM UCN-01 for 72 h and at 500 nM for 30 h, RB-transfected sublines showed an accumulation of cells in G0–G1 and a loss of S-phase cells to a greater degree than DU-145 parent cells.

 


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  		Fig. 7. Distribution of cell cycle phases for DU145, B5, and DU1.1 untreated (0 h) and after treatment with UCN-01 at 100 nM for 72 h and at 500 nM for 30 h. The percentages of cells in each cell cycle phase (Table 2)Citation are depicted by G1 (black), S (white), and G2 (gray).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lung cancer is now the most common cause of cancer-related death in both men and women. Because most patients present with advanced stage disease, improved systemic therapy is essential. UCN-01 represents a new class of anticancer agents with a novel therapeutic target: the cell cycle. Disruption of cell cycle regulation is a common motif in lung cancer. In NSCLC cell lines, abnormal RB is found at a frequency of 15%, whereas p16CDKN2 alterations are more common, estimated at 45% (11 , 12) . In contrast, RB abnormalities are common in SCLC, reported as high as 88%, whereas p16CDKN2 abnormalities occur rarely (11 , 12) . In both types of cancer, p53 alterations are relatively common, with frequencies of ~45% for NSCLC and as high as 80–100% for SCLC (35) . Reported here are investigations of the molecular mechanisms of UCN-01 response using an experimental model of human NSCLC lines with differential abnormalities in these cell cycle regulatory genes.

This report demonstrates that response to UCN-01 is associated with wild-type RB status in NSCLC cells and is characterized by G1 checkpoint arrest. Evidence supporting the role of wild-type RB status as a determinant of UCN-01 response includes the induction of p21CDKN1 expression and the modulation of the phosphorylation state of Rb, a possible mechanism of G1 arrest.

To further test the role of RB in the response to UCN-01, we used two established human isogenic models of RB expression, one a bladder carcinoma line and the other a prostate carcinoma, because of the absence of a NSCLC model. In the bladder cancer model, the RB-expressing subline showed Rb hypophosphorylation and G1 arrest at 100 nM treatment, in contrast to the parent line. In the prostate cancer model, the wild-type RB-expressing sublines showed greater G1 accumulation and a greater diminishment of S-phase cells when treated with UCN-01. Thus, these data also support the hypothesis that RB status is a determinant of response to UCN-01. However, the limited G1 arrest seen in both parent lines of these isogenic models indicates that RB is not the sole determinant of G1 arrest in response to UCN-01.

Previously, Seynaeve et al. (15) reported differential sensitivity to UCN-01 treatment in a series of breast cancer cell lines, with some lines exhibiting near complete, irreversible growth arrest, whereas others showed a more limited response. On the basis of our findings, these results may be clarified by the observation that the MDA-MB-468 cell line, which was the least responsive, is RB-mutant (36) . The association of UCN-01 response with wild-type RB is further supported by similar results reported by our group with staurosporine, a closely related compound (26 , 37 , 38) . RB was required for staurosporine-induced G1 arrest in human bladder carcinoma 5637 cells as demonstrated by the RB5 subline.

Additional conclusions from this study are as follows: (a) because p16CDKN2-null cells (A549) were strongly inhibited by UCN-01 and no increase in expression of this Cdk inhibitor was observed, we conclude that p16CDKN2 is not required for UCN-01-induced growth arrest; and (b) the finding that UCN-01 induced p21CDKN1 expression and growth arrest in p53-null cells (Calu1) suggests that UCN-01 functions, at least in part, through the p53-independent induction of p21CDKN1. These observations do not rule out the possibility that UCN-01 may operate additionally through a p53-dependent pathway; p53-null Calu1 cells were growth inhibited less than the wild-type p53 cell lines T3891 and A549. However, this difference was not statistically significant. Recently, Husain et al. (39) showed that the activity of UCN-01 as a single agent was independent of p53 status in a human ovarian cancer model. However, their p53 transfection experiments suggested that G1 arrest in response to UCN-01 was p53 dependent. In contrast, our data in a p53-null NSCLC cell line implicate the p21CDKN1 and RB genes as playing key roles in the G1 arrest.

UCN-01 is also of therapeutic interest due to its ability to potentiate the anticancer activity of chemotherapy and radiation. Others have reported that potentiation of DNA-damaging agents, such as radiation and cisplatin, by UCN-01 is greater in cells with disrupted p53 (18 , 19) . In our studies, UCN-01 as a single agent demonstrated activity in cell lines both wild-type and null for p53. The ability of UCN-01 to sensitize cells to cisplatin or radiation appears to result from an abrogation of the DNA damage-induced G2 checkpoint arrest (18 , 19) . In contrast, the mechanism of anticancer action of UCN-01 as a single agent is clearly different, where G1 arrest is a predominant feature.

In conclusion, our findings suggest that the molecular profile of key cell cycle regulatory genes in human cancers may predict responsiveness to new antineoplastic agents. Because UCN-01 is now entering early clinical trials in cancer patients, additional investigations designed to clarify further the molecular mechanisms of action of this novel therapeutic agent are warranted.


   ACKNOWLEDGMENTS
 
We thank Drs. Arline Deitch and Kayoko Nishi for advice on these studies. We also thank Dr. Edward A. Sausville for providing the UCN-01 and Dr. David W. Goodrich for providing the 5637 cells and the RB-expressing subline RB5. Additionally, our thanks go to Jared T. Muenzer, Jeff Yun, Mahesh Patel, and Andrew Hufton for assistance with cell culturing and Western blotting.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by Grants CA62505, CA63265, CA/AG79912, and CA75178 from the NIH and grants from the Veterans Administration and the Sheeter Foundation. Back

2 Present address: Greenebaum Cancer Center, University of Maryland Medicine, Baltimore, MD. Back

3 To whom requests for reprints should be addressed, at Division of Hematology and Oncology, UC Davis Cancer Center, 4501 X Street, Sacramento, CA 95817. Phone: (916) 734-8614; Fax: (916) 734-2361. Back

4 The abbreviations used are: Rb, retinoblastoma; Cdk, cyclin-dependent kinase; NSCLC, non-small cell lung carcinoma; UCN-01, 7-hydroxystaurosporine; PKC, protein kinase C; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PI, propidium iodide. Back

Received 1/12/99; revised 5/10/99; accepted 5/24/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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