Date of Award
Doctor of Philosophy
The ATP-binding cassette (ABC) superfamily of membrane transporters use energy derived from ATP to eliminate a variety of exogenous and endogenous compounds from cells including anti cancer and anti viral drugs, metals steroids, bilirubin, cAMP, cGMP, leukotrienes , prostaglandins. The multi drug resistance associated protein family (MRP/ABBC) has been acknowledged as a major player involved in multi drug resistance (MDR), in which cancers stop responding a wide array of structurally and functionally unrelated chemotherapy drugs. Cancer is the second leading cause of death in adults in the United States. Although there have been great strides made the treatment of cancers over the last 50 years, there are still 1.5 million new cases and 0.5 million deaths a year in the US. The death rates for several cancers including cervical cancer and stomach cancer in both men and women are a third of what they were 40 years ago. However, for melanoma, esophageal, pancreatic and liver cancer death rates have actually increased. There are various ongoing lines of research attempting to develop new therapies and strategies to increases the efficacy of chemotherapy. One approach which has met with varying degrees of success has been the manipulation of drug transporters involved in MDR. Inhibiting the MRPs and there by reversing the MDR phenotype would allow the use of existing chemotherapy drugs and treatment regimes at lower doses , thereby decreasing the magnitude of the side effects experienced patient undergoing current treatment regimes. However the endogenous function of MRP1 has not been well characterized, therefore inhibiting MRP1 may have unforeseen consequences. We there for decided to look at an Mrp1 knock out mouse to determine any potential compensatory changes that might result from the loss of Mrp1. Work has previously been done examining changes in the liver of the Mrp1-/- mouse, we therefore looked at extra hepatic tissues. We discovered increased renal cytochrome P405 activity in the Mrp1 -/- mice. Phase I P450 activity was altered in the testes as well, with a reduction of Cyp2c11 activity and an induction of Cyp17 activity. In addition to alterations in phase I activity, Mrp1-/- mice had increased expression of the phase II conjugation enzyme sulfotransferase 1a1 in the small intestine, and a reduced expression in the lungs. Interestingly, we fund that Mrp2 and Mrp4 expression was reduced in the lungs and Mrp2 was reduced in the kidneys of the Mrp1-/- mice. We also sought to determine any modifications that may have resulted from the loss of Mrp1 in the testes. As Mrp1 is highly expressed in the testes and is thought to be involved in maintaining steroid homeostasis by expelling excess estrogen from the testes, we examined both testicular and serum steroid levels. We discovered that the loss of Mrp1 leads to decreased steroid levels in both the testes and serum. There is a significant reduction of androstenedione, estradiol, testosterone, and dehydroepiandrosterone (DHEA) in the testes of Mrp1 knockout mice. In order to resolve the mechanisms involved in the reduction of serum steroid levels we examined testicular steroid biosynthesi pathways. We found no alterations in sulfotransferase or glucuronosyltransferase enzyme activity. So it appears that in order to circumvent the buildup of excess estrogen in the testes, steroid production is attenuated, resulting in lower androsteindione, testosterone, and estradiol in the testes. The decrease in circulating testosterone is in all likelihood responsible for the alterations seen in the kidney, ling and small intestines.
We also examined Mrp1-/- mice for alterations in drug disposition using tritiated vincristine for our test compound. The loss of Mrp1and the resulting alteration in Mrp expression leads to significant build up of vincristine in the kidney, small intestine, colon, and epididymis. This buildup of vincristine is almost certainly an attempt to eliminate the vincristine and results from the variation in transporter expression along the GI track and the kidney.
Mrp1 is also expressed in dendritic cells(DC) and considering Mrp1 and Mrp4 have been implicated in DC function and a required for proper DC trafficking we decided to determine if either of the transporters are required for efficient DC maturation and activation. By using the Mrp1-/- mouse model, and a specific Mrp4 inhibitor, we determined potential role Mrp1 and Mrp4 play DC function. The loss of either Mrp1 and/or Mrp4 hindered DC ability to produce IL12 when stimulated by LPS. If either of the transporters are lost during maturation, upon activation by LPS, the DCs have reduced induction of activation markers CD54, CD 80, CD86 and MHCII, as well as reduced IL12 production. As the ultimate test of DC function is activation of T cells, after inhibition of transporter activity during maturation, we examined how effectively the DCs could activate T cells. Without functioning transporters, the ability DCs to effectively activate T cells is severely crippled.
Received from ProQuest
Jeffrey Charles Sivils
Sivils, Jeffrey Charles, "Effects Of The Loss Of Multidrug Resistance Associated Protein 1 On Steroid Homeostasis, Dendritic Cell Function And Compensatory Mechanisms" (2010). Open Access Theses & Dissertations. 2785.