Although the coincidental amplification and accompanying overexpression of bystander genes that neighbor oncogene targets occur frequently during the development of human tumors, little has been done to investigate the functional or biological consequences of amplified bystander gene overexpression. LANCL2 (LANC-like 2) is a bystander gene that is coamplified and overexpressed with epidermal growth factor receptor in approximately 20% of all glioblastomas. This gene has also been designated as Testis Adriamycin Sensitivity Protein because it is most highly expressed in testis and its expression has been noted to increase cellular sensitivity to Adriamycin. Because of the latter association, we have examined potential relationships between LANCL2 and the expression of multidrug-resistance (MDR)1, as well as its cognate protein, P-glycoprotein (P-gp), because elevated expression of P-gp is known to increase cell resistance to many cytotoxic drugs, including Adriamycin. Using the Dx5 derivative of MES-SA cells in which P-gp is overexpressed, we show that the level of endogenous P-gp decreases with increased expression of exogenous LanCl-2 and that cells with reduced P-gp show increased sensitivity to Adriamycin. Results from reverse transcription-PCR and MDR1 promoter activity analyses suggest that LanCl-2 transcriptionally suppresses MDR1, and this interpretation of LanCl-2 function is consistent with results from immunofluorescence analysis, which shows that LanCl-2 resides in the nucleus, as well as at the plasma membrane. With respect to this study, our data indicate that LanCl-2 increases cellular sensitivity to Adriamycin by decreasing the expression of P-gp, but more generally, these results indicate that the identification of bystander gene amplification in human tumors can have clinical implications.