Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines

Abstract

Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in alpha4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and alpha4-low phenotype. Whether VLA-4-mediated FN adhesion offers a survival advantage over VLA-5-mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.

Fig. 1

8226/S myeloma cells adhered to FN have a survival advantage over nonadhered cells following acute doxorubicin exposure (A) but not following melphalan exposure (B) in cell growth based cytotoxicity assays. FN-adhered cells (---) were bound to FN-coated plates 24 hours before 1-hour drug exposure and control cells were grown in suspension (—). Response to doxorubicin was 12.6-fold lower in FN-adhered cells compared to nonadhered controls (IC50 values for adhered and nonadhered cells were of 4.85 × 10⁻⁷ mol/L and 8.5 × 10⁻⁸ mol/L, respectively). Data points are presented as cell viability determined by MTT cytotoxicity assay compared with untreated controls. Graphs are representative experiments that were repeated three times in replicates of four.

Fig. 2

Annexin V stained FN-adhered myeloma cells have a lower apoptotic fraction compared to nonadhered cells following acute drug exposure. 8226/S myeloma cells were exposed to 1 μmol/L doxorubicin for 1 hour (A) or 50 μmol/L melphalan for 24 hours (B), stained by Annexin V 24 hours later, then analyzed by flow cytometry. Histograms are adjusted for background staining in untreated cells, bars are the SD of three different experiments; *, P < .05.

Fig. 3

RNA levels of Bcl-2 family members are unchanged following FN adhesion. Drug sensitive 8226/S cells were adhered to FN-coated plates or grown in suspension for 24 hours after which total RNA was collected and analyzed by RNase protection. Expression levels were normalized to the housekeeping genes GAPDH and L32.

Fig. 4

Intracellular doxorubicin concentration is unaffected by culturing cells on plastic, BSA, or FN. Following a 24-hour incubation on each surface, 10 μmol/L doxorubicin was added to each well for 1 hour and cells were analyzed for drug accumulation differences by flow cytometry. Bars are the SD of n = 6 from two independent experiments.

Fig. 5

Phenotypic analysis of 8226 cell surface FN receptor expression by flow cytometry. Integrin subunit expression by drug-sensitive (8226/S), melphalan-resistant (8226/LR5), and doxorubicin-resistant (8226/DOX6) cell lines were analyzed using MoAbs for α₄, α₅, β₁, and β₇. Cells were incubated with an integrin-specific MoAb (—) or with irrelevant control Ab (---), followed by incubation with FITC-conjugated secondary Ab. Ten thousand events were analyzed for each sample using a FACScan machine (Becton-Dickinson); histograms are representative of three different experiments.

Fig. 6

(A) Drug resistance is associated with α₄ expression in melphalan-resistant (8226/LR5) and revertant (LR5ood) cell lines. 8226/LR5 were maintained in 5 × 10⁻⁵ mmol/L melphalan (LPAM) and LR5ood were maintained out of drug for 20 weeks. α₄ expression was measured by flow cytometry and drug resistance was measured by MTT cytotoxicity analysis. Resistance values are reported as the IC50 dose of LPAM relative to 8226/S. α₄ expression levels and melphalan resistance levels of 8226/LR5 were found to be higher than 8226/S (P < .05). α₄ expression and melphalan resistance of LR5ood were found to be equal to those of the 8226/S parent line. Bars are the SD of three different experiments. (B) Drug resistance is associated with α₄ expression in doxorubicin-resistant (8226/DOX6) and revertant (DOX6ood) cell lines. 8226/DOX6 were maintained in 6 × 10⁻⁸ mol/L doxorubicin and DOX6ood were maintained out of drug for 20 weeks. α₄ expression was measured by flow cytometry and drug resistance was measured by MTT cytotoxicity analysis. Resistance values are reported as the IC50 dose of doxorubicin relative to 8226/S. α₄ expression levels and doxorubicin resistance levels of 8226/DOX6 were found to be higher than 8226/S (P < .05). α₄ expression and doxorubicin resistance of DOX6ood were found to be equal to those of the 8226/S parent line. Bars are the SD of three different experiments.

Fig. 7

Contribution of α₄ and α₅ integrin subunits to FN adhesion. Drug-sensitive (8226/S), melphalan-resistant (8226/LR5), and doxorubicin-resistant (8226/DOX6) were adhered to FN-coated wells (horizontal striped bars) for 1 hour. In order to determine percentage binding due to α₄ and α₅, some cells were pre-incubated with α₄ function blocking Ab P4G9 (hatched bars) or α₅ function blocking Ab P1D6 (vertical striped bars) for 15 minutes before application to wells. FN adhesion by 8226/S was found to be mediated equally by α₄ and α₅, while FN adhesion for both drug-resistant cell lines was mediatedonly by α₄ (P < .05), as determined by complete inhibition of adherence using the α₄ blocking Ab. Total FN adhesion mediated by α₄ was also higher in drug-resistant lines compared with drug-sensitive 8226/S (P < .05). Values shown are the percentage of total cells applied to each well corrected for nonspecific adhesion to BSA-coated wells. Bars are the SD of n = 6 from three different experiments.