The Effects Of Ethidium Bromide Induced Loss Of Mitochondrial DNA On Cellular Function And Ultrastructure In Human Breast Cancer Cell Line T47D

Breast cancer is one of the most common maligancies in female across the world. In western countries, breast cancer ranks first among all the female cancers; whileas in china, although breast cancer is still of low incidence rate in general, it has dramastically increased about 30% in the past 10 years. In addition, in most big cities such as Beijing, Shanghai&Tianjin, breast cancer has also jumped to the first in the female maligancy incidence-ranking list.In recent years, the correlation between alterations in mitochondrial DNA (mtDNA) and various sorts of solid tumors including breast tumor has been broadly investigated. In order to further explore the potential function of mtDNA mutation in the etiology and pathogenesis of breast tumor and elucidate the molecular genetic mechanism of nucleo-mitochondrial interaction, it is useful and necessary to isolate a special cell model that completely lacks mtDNA, denoted as mtDNA-less or rho-zero (ρ~0) cell line. The ρ~0 cell line allows us to manipulate the mtDNA complement of a cell, move mitochondria from one cellular environment to another, or introduce new genes into mitochondria and could work as an excellent research platform for better understanding the potential contributions of mitochondrial genome in human maligancies.The primary objective of our project is to establish a viable breast cancer T47D cell mutant depleted of mtDNA by applying low-dose ethidium bromide (EtBr), a well-known mtDNA replication inhibitor, and then explores the effects of depleting mtDNA on cellular processes and ultrastructure in comparision with its parental cell line. There are mainly two parts in our research:Part Ⅰ : To develop ρ~0 cells, T47D cells were chronically exposed to 50ng/ml EtBr in medium supplemented with pyruvate and uridine. After approximately 32 days, the EtBr treatment was discontinued and ρ~0 cells were ready for further identification. In our T47D ρ~0 cells, DNA assays(southern dot blots and real-time PCR) confirmed the absense of mtDNA and selective ρ~0 test medium analysis verified its autrophic growth characteristics for uridine. The T47D ρ~0 cell derivative can steadily grow and divide in RPMI 1640 supplemented with 15% FBS, sufficient uridine and pyruvate.