Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells

The p16 tumor suppressor gene is frequently inactivated in human cancer tissues and cell lines. We previously reported that wild-type p16 expression from an adenovirus vector (Adv/p16) induced p53-dependent apoptotic cell death in non-small cell lung cancer (NSCLC) cell lines. Here we show the potential mechanism of apoptosis induced by Adv/p16 infection. Infection of human NSCLC cell line A549, which carries the wild-type p53 gene, with Adv/p16 resulted in activation of caspase-3, accompanied by the cleavage of its substrate poly (ADP-ribose) polymerase (PARP), on day 3 of infection. The retinoblastoma (Rb) cell cycle regulator protein was also cleaved after activation of caspase-3; when the levels of Rb significantly diminished, apoptosis began. When A549 cells were pretreated with the caspase-inhibitory peptide N-acetyl-asp-Glu-Val-Asp-CHO (aldehyde) (Ac-DEVD-CHO), Adv/p16-mediated apoptosis and Rb cleavage were greatly inhibited. Furthermore, MDM2, a negative regulator of p53 expression was upregulated 3 days after Adv/p16 infection, and MDM2 was subsequently cleaved by caspase-3; MDM2 cleavage was inhibited by Ac-DEVD-CHO treatment. These data implied that cleavage of Rb, in addition to activation of caspase-3, represented a mechanism by which Adv/p16 induced apoptotic cell death in human NSCLC cells. Our results support the clinical relevance of Adv/p16 as a treatment for p16-null human NSCLC that express wild-type p53.     

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.     

Analysis of K-ras, p53, bcl-2 and Rb expression in non-small cell lung cancer cell lines

Six non-small cell lung cancer (NSCLC) cell lines (A-549, Ca-Lu-6, SK-Lu-1, Ca-Lu-1, SK-Mes-1 and LX-1) were studied to assess the presence of multiple concomitant alterations of different oncogenes (K-ras, bcl-2) and tumor suppressor genes (p53, Rb) in NSCLC. K-ras (exon 1) and p53 (exons 5-8) gene mutations were determined via a PCR-based-DGGE (Denaturing Gradient Gel Electro-phoresis) and by sequencing approach. Different mutations were found in the Ist exon of K-ras gene in 5 of 6 cell lines examined. Five of six cell lines contained K-ras mutations at codon 12 (A-549, SK-Lu-1, LX-1) or codon 13 (SK-Mes-1, Ca-Lu-1). In addition, 5 of 6 cell lines showed p53 mutations of exon 8 (SK-Mes-1, Ca-Lu-1 cod. 280; LX-1 cod. 273) or exon 6 (Ca-Lu-6 cod. 196; SK-Lu-1 cod. 193). In 4 of these cell lines, p53 protein nuclear expression was also confirmed with DO-7 mAb immunocytochemistry. Expression of cytoplasmic bcl-2 protein, by anti-bcl-2 mAb flow cytometric analysis, was found in A-549, Ca-Lu-1, SK-Lu-1, SK-Mes-1 cell lines. In contrast, RT-PCR analysis of Rb gene could not identify any change in the cell lines examined. In conclusion, most NSCLC cell lines tested displayed concomitant multiple oncogene/tumor suppressor gene alterations.     

Differential post-translational modification of the tumour suppressor proteins Rb and p53 modulate the rates of radiation-induced apoptosis in vivo.

Ionizing radiation induces p53-dependent apoptosis in the spleen, providing a model system to study p53 regulated events in a normal cell type. We have developed an in vivo model that identifies genetic differences in the regulation of p53-mediated apoptosis and addresses whether altered post-translational events in the p53-p21/Rb axis modulate the sensitivity of cells to radiation-induced cell death in vivo. Splenocytes from mice with distinct genetic backgrounds (DBA/2 and C57BL/6) exhibit differences in the rate of apoptosis. Whilst no obvious strain differences in protein levels of Bcl-2 or the cyclin-CDKs were observed, early post-translational regulatory events in the p53-p21/Rb axis showed striking differences in the two mouse strains. Cells from C57BL/6 animals undergo more rapid apoptosis after irradiation resulting from elevated levels and rapid induction of p53, pronounced Rb-cleavage, and the absence of a sustained induction of p21. In contrast, cells from DBA/2 animals have a reduced rate of apoptosis following irradiation with elevated levels of hyperphosphorylated Rb and a sustained induction of the p21 protein that is coincident with the C-terminal phosphorylation of p53. These data suggest that quantitative differences in the level of p21 protein can affect the rate of apoptosis in vivo, consistent with the view that p21 is an anti-apoptotic effector of p53. However, striking differences in the Rb protein-caspase cleavage or hyperphosphorylation-in the same cell type, but in different genetic backgrounds, demonstrates that p53-dependent apoptosis can be modulated in vivo by genetic factors that impinge upon the pro- or anti-apoptotic potential of Rb. In addition, we show that Rb cleavage is p53-dependent and that its phosphorylation status can be uncoupled from p21 expression. This study highlights the possibility that genetic factors can be identified that affect differential sensitivity of cells to ionizing radiation in vivo.     

Abnormal expression pattern of the ASPP family of proteins in human non-small cell lung cancer and regulatory functions on apoptosis through p53 by iASPP

The p53 protein is one of the best-known tumor suppressors. Recently discovered ASPP1 and ASPP2 are specific activators and iASPP is an inhibitor of p53. In the present study, we found that of 37 NSCLC patients, p53 alterations were detected in 20 tumors (54.1%), the mRNA expression of ASPP1 and ASPP2 was frequently dowregulated in tumor tissues, and this decreased significantly in samples expressing wild-type p53. The expression of ASPP1 and ASPP2 was downregulated and that of iASPP was upregulated in two NSCLC cell lines (the NCI-H157 cell line with altered p53 and the A549 cell line with wild-type p53). The NCI-H157 cell with higher ASPP1 and ASPP2 levels was more sensitive to cisplatin than the A549 cells with lower ASPP1 and ASPP2 levels. Downregulation of iASPP by siRNA stimulated apoptosis through p53 in two NSCLC cell lines. These findings provide new insights into the molecular mechanisms of action of the ASPP family in NSCLC and may have potent therapeutic applications.     

p53 enhances gefitinib-induced growth inhibition and apoptosis by regulation of Fas in non-small cell lung cancer

Treatment with gefitinib, a specific inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), has resulted in dramatic responses in some patients with non-small cell lung cancer (NSCLC). Most patients who respond to gefitinib have EGFR-TK mutations; however, >10% of patients with EGFR-TK mutations do not respond. Similarly, some patients without EGFR-TK mutations respond to this drug, suggesting that other factors determine sensitivity to gefitinib. Aberrations of the tumor suppressor gene p53 are frequently associated with drug resistance. In this study, we investigated the role of p53 in growth-inhibitory and apoptotic effects of gefitinib in the human NSCLC cell lines NCI-H1299 and A549, which have no EGFR-TK mutations. NCI-H1299 cells, which had a p53-null genotype, were more resistant to gefitinib compared with A549 cells, which were wild-type p53 (IC(50), 40 micromol/L in NCI-H1299 and 5 micromol/L in A549). Treatment of A549 with gefitinib resulted in the translocation of p53 from cytosol to nucleus and the up-regulation of Fas, which was localized to the plasma membrane. In the stable H1299 cell line with tetracycline-inducible p53 expression, induced p53 enhanced growth inhibition and apoptosis by gefitinib through the up-regulation of Fas and restoration of caspase activation. A caspase inhibitor, Z-VAD-fmk, reduced these effects. Conversely, inhibition of p53 using antisense oligonucleotide in A549 caused a significant decrease in apoptosis by gefitinib and down-regulation of Fas under the same conditions. In conclusion, p53 may play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC.     

Role of p53 in the induction of cyclooxygenase-2 by cisplatin or paclitaxel in non-small cell lung cancer cell lines

Non-small cell lung Cancer (NSCLC) is extremely resistant to chemotherapeutic agents, such as cisplatin. High expression of the inflammatory enzyme Cyclooxygenase-2 (COX-2) has been shown to inhibit chemotherapy-induced apoptosis, but little is known about COX-2 regulation upon drug treatment. Recent data indicate the tumor suppressor protein p53 as an important regulator of COX-2. Therefore, TP53 status could change tumor sensitivity to chemotherapy through induction of the anti-apoptotic protein COX-2. The main objective of this work was to analyze the effect of chemotherapy on the expression of COX-2, according to TP53 status. We report herein that lung cancer cell lines expressing wild-type p53, when exposed to cisplatin treatment, induced COX-2 (mRNA and protein), with concurrent synthesis of prostaglandins (PGE₂). In contrast, COX-2 expression was not changed after cisplatin treatment of cells containing an inactive form of p53. Further, after silencing of wild-type p53 expressed in A549 cells by RNA interference, cisplatin was no longer able to induce COX-2 expression. Therefore, we suggest that induction of COX-2 by cisplatin in NSCLC cell lines is dependent on p53. For paclitaxel treatment, an increase in COX-2 mRNA expression was observed in H460 and A549 (wild-type p53 cell lines). Moreover, paclitaxel treatment increased COX-2 expression in ACC-LC-319 cell lines (p53 null), showing a p53-independent effect. These data may have therapeutic implications in the selection of patients and strategy for future COX-2 inhibition trials.     

The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function

VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with > or =4N DNA content, followed by cell death. Here, we define the role of p53 and p21(Waf1/Cip1) in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21(Waf1/Cip1) and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21(Waf1/Cip1) within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21(Waf1/Cip1) is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21(Waf1/Cip1) expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21(Waf1/Cip1) in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21(Waf1/Cip1)-dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis.     

Molecular determinants of cell death induction following adenovirus-mediated gene transfer of wild-type p53 in prostate cancer cells

Adenoviral vectors expressing wild-type p53 (Ad-p53) induce apoptosis in different types of cancer cells. The therapeutic utility of Ad-p53 is now being evaluated in prostate-cancer patients. Bcl-2 is frequently expressed by prostate-cancer cells and has previously been shown to inhibit p53-mediated cell death following genotoxic stress. We studied the impact of bcl-2 on Ad-p53-induced cell death in human prostate-cancer cells. Human prostate-cancer cell lines LNCaP (p53 wt) and PC3 (p53 mut) were stably transfected with bcl-2. After p53 transduction, cell viability, apoptosis induction and modulation of specific apoptosis-regulatory proteins were assessed. LNCaP vector control and bcl-2-expressing cells underwent similar decreases in viability associated with apoptosis induction following Ad-p53 infection. Increased bcl-2 expression provided significant protection to PC3 cells transduced with Ad-p53. These findings are correlated with modulations in bax, bcl-2, bcl-x(L) and p21 protein levels. These data suggest that Ad-p53 may be useful in the treatment of some prostate cancers.     

In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.

Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.     

Apoptosis induced by adenovirus-mediated p53 gene transfer in human glioma correlates with site-specific phosphorylation

Therapeutic replacement of the p53 gene using an adenovirus vector (Ad-p53) may be an effective alternative to conventional therapies for the treatment of glioma. We have previously demonstrated that the introduction of Ad-p53 into glioma cells containing mutant p53 induces apoptosis, whereas glioma cells containing wild-type p53 are resistant. However, Ad-p53 will enhance the radiosensitivity of wild-type p53 glioma cells by increasing their tendency for apoptosis. The mechanism underlying these different responses to Ad-p53 has not been elucidated to date. Because phosphorylation of p53 at serines 15, 20, and 392 may play a role in regulating p53-mediated apoptotic activity, we determined the phosphorylation status of exogenous p53 in mutant and wild-type gliomas after Ad-p53 transfer. Monolayer cultures of glioma cell lines expressing mutant p53 (U251 and U373) or wild-type p53 (U87 and D54) were infected with Ad-p53 and analyzed by Western blotting. High levels of exogenous p53 were detected in both cell lines after Ad-p53 transfer. However, only apoptotic mutant p53 cells expressed high levels of phospho-Ser15-p53 and phospho-Ser20-p53. The levels of phospho-Ser15-p53 and phospho-Ser20-p53 were very low in wild-type p53 cells after Ad-p53 infection alone. When wild-type p53 glioma cells were exposed to radiation after Ad-p53 infection, phospho-Ser15-p53 and phospho-Ser20-p53 were detected at high levels, and the cells subsequently underwent apoptosis; no change in serine 392 was detected. The induction of apoptosis and the expression of phospho-Ser15 and phospho-Ser20 in these cells were also enhanced by the combination of Ad-p53 and other DNA-damaging agents such as cisplatin and bichloroethyl nitrosourea. Furthermore, the expression of phospho-Ser15-p53 and phospho-Ser20-p53 correlated with the amount of apoptosis; the apoptotic activity of p53 in glioma cells was partially inhibited by a mutation of p53 at serine 15. These results suggest that phosphorylation of p53 at serine 15 and serine 20 is critical for apoptosis induction in p53 gene therapy for gliomas.     

p21Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistance in a camptothecin-selected human lung-cancer cell line

The realization that chemotherapeutic agents induce apoptosis raises the concern that tumors resistant to chemotherapy are unable to initiate the apoptotic program. In the present study, we examined the apoptosis-resistance mechanism of a multidrug-resistant cell line, A549/CPT, which was established from the human lung-cancer cell line A549 by in vitro selection with gradually increased camptothecin (CPT) concentrations. We found that A549/CPT cells were resistant to anti-cancer drug-induced apoptosis in which caspase-3-like protease activity was attenuated remarkably, compared with parental A549 cells. We observed 2 mechanisms associated with apoptosis resistance in A549/CPT cells: over-expression of anti-apoptotic bcl-2 and elevated expression of p21Waf1/Cip1. Transfection of either bcl-2 or p21Waf1/Cip1 cDNA into parental A549 cells resulted in resistance to apoptosis. Furthermore, the co-treatment of p21Waf1/Cip1 and bcl-2 anti-sense oligodeoxy-nucleotides restored drug susceptibility in A549/CPT cells more effectively than either one of them alone. These results indicate that co-induction of bcl-2 and p21Waf1/Cip1 in A549/CPT cells may be involved in acquired drug resistance by inhibiting caspase-mediated apoptosis. Agents aimed at preventing both bcl-2 and p21Waf1/Cip1 expression may increase the efficiency of chemotherapy.     

Increased radiation-induced apoptosis and altered cell cycle progression of human lung cancer cell lines by antisense oligodeoxynucleotides targeting p53 and p21(WAF1/CIP1)

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21(WAF1/CIP1). Delayed apoptosis (35.7+/-4.2% in H460, 1.2+/-0.4% in A549 and 72.2+/-6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96 hours by 17.9+/-8.5 and 9.1+/-3.3% to 53.6+/-7.4 and 10.8+/-2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8+/-5.2 and 31.6+/-7.3% to 59.4+/-3.1 and 32.8+/-7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21(WAF1/CIP1) mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.     

Changes in p21WAF1, pRb, Mdm-2, Bax and Bcl-2 expression in cervical cancer cell lines transfected with a p53 expressing adenovirus

The aim of this study was to provide some insights into the molecular mechanisms involved in p53-dependent apoptosis and growth arrest. Changes in the levels of p53 protein and proteins regulated by p53 were studied in relation to events of the cell cycle and apoptosis in cervical cancer cell lines upon transfection with a p53 expressing adenovirus (Ad5-p53). The post-transfection level of p53 protein in SiHa cells was found to be unchanged during the 24-48 h period. In contrast, the level of p21WAF1 protein was shown to increase to its highest level at 24 h, and decreased gradually up to 48 h after the Ad5-p53 transfection. We further noted that the increase of p21WAF1 was accompanied by G1 arrest at 24 h and the decrease of p21WAF1 was associated with apoptosis at 36-48 h after transfection. An anti-p21WAF1 antibody cross-reactive protein band of approximately 14 kDa was observed in HeLa and C-33A cells when these cells were committed to apoptosis upon Ad5-p53 transfection. In SiHa cells, phosphorylation of pRb was inhibited during the early stage of Ad5-p53 transfection. This was followed by the cleavage of pRb. However, Ad5-p53 transfection did not change the levels of Bax and Bcl-2 proteins. Our results suggested that, Bax and Bcl-2 may not be important for the apoptosis of these cells, whereas cleavage of Rb, and the decrease of p21WAF1 could play important roles in p53-dependent apoptosis.     

Adenovirus-mediated transfer of wild-type p53 gene results in apoptosis or growth arrest in human cultured gastric carcinoma cells.

We examined the susceptibility of six human gastric carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). AxCA-p53 infection at a muliplicity of infection (MOI) of 50 resulted in apoptotic cell death (MKN-1 cells), growth arrest (MKN-45, MKN-74 and KATO-III cells), or non-effectiveness (TMK-1 and OCUM-2M cells). Western blot analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 in all the cell lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in MKN-1, but not in the other cell lines. These results suggest that the apoptotic pathway (dependence on the expressions of bcl-2 family proteins) dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCA-p53. Thus, the bcl-2 family might play a crucial role in p53-mediated growth arrest and apoptosis in human gastric carcinoma cells.     

Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. In order to understand the molecular alterations leading to heterogeneous cisplatin sensitivity and apoptosis inducibility in NSCLC cells, we analyzed various apoptotic pathways, including the activation of caspase-8, -9 and -3, the release of cytochrome c from mitochondria and the expression levels of pro- and anti-apoptotic proteins such as Bax, Bad, Bcl-2, Bcl-xL, Fas and p53 using heterogeneously apoptosis-sensitive cells (Ma-10, Ma-31 and Ma-46). Cisplatin treatment induced the activation of caspase-8, -9 and -3 and the release of cytochrome c in apoptosis-sensitive Ma-46. The expression of Bcl-xL was the highest and p53 was not expressed in apoptosis-resistant Ma-31, and Fas was not expressed in Ma-46. These expression levels were not correlated with the apoptosis inducibility of the three cell lines. These results suggest that blockage of the apoptotic signal from mitochondria is responsible for apoptosis resistance in NSCLC cell lines. Our findings also indicate that anti-apoptotic Bcl-xL and pro-apoptotic p53 are necessary but not sufficient for resistance to cisplatin-induced apoptosis in NSCLC cells.     

Interactions of gemcitabine, carboplatin and paclitaxel in molecularly defined non-small-cell lung cancer cell lines

PURPOSE: To evaluate in vitro interactions of carboplatin, gemcitabine and paclitaxel in molecularly defined non-small-cell lung cancer lines. MATERIALS AND METHODS: Three NSCLC lines, A549 (p16-,p53 wt, Rb wt), Calu-1 (p16-, p53-, Rb+) and H596 (p16 wt, p53 mut, Rb-) were utilized. Cells were exposed to carboplatin, gemcitabine and paclitaxel as individual drugs and in two- and three-drug combinations with various sequences of administration. Cytotoxicity was assessed with the MTT assay. Interactions between the drugs (additive, synergistic and antagonistic) were evaluated by median effect analysis. RESULTS: Gemcitabine and carboplatin were synergistic in all three cell lines. In the A549 line, this synergy was most pronounced when gemcitabine preceded carboplatin. For three-drug combinations, paclitaxel was synergistic with gemcitabine and carboplatin regardless of sequence of administration. CONCLUSIONS: In vitro modeling of gemcitabine and carboplatin as well as gemcitabine/carboplatin and paclitaxel demonstrates synergistic interaction regardless of p16, p53, or Rb status.