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Type: Thesis
Title: Regulation of the 24 - hydroxylase gene promoter by 1,25 - dihydroxyvitamin D3 and chemotherapeutics drugs
Author: Tan, Cheng Ta Joseph
Issue Date: 2005
School/Discipline: School of Paediatrics and Reproductive Health
Abstract: Chemotherapy in childhood cancer patients is associated with reduced bone density that can result in osteoporotic fracture in survivors. A significant proportion of paediatric patients experience a reduction in plasma 25 - hydroxyvitamin D3 [ 25 ( OH ) D3 ] and 1,25 - dihydroxyvitamin D3 [ 1,25 ( OH ) 2D3 ] levels during treatment, the basis of which is unknown. A balance between the bioactivation and degradation of 1,25 ( OH ) 2D3 is responsible for maintaining homoeostatic levels of 1,25 ( OH ) 2D3 at the correct set - point. Whereas the cytochrome P450 enzyme, CYP27B1 ( 25 - hydroxyvitamin D3 1 α - hydroxylase ), catalyses the hydroxylation of the precursor 25 ( OH ) D3 to generate 1,25 ( OH ) 2D3, catabolic inactivation and cleavage of 1,25 ( OH ) 2D3 is achieved by the mitochondrial cytochrome P450 enzyme, 25 - hydroxyvitamin D3 24 - hydroxylase ( CYP24 ), which is highly expressed in bone and kidney cells. Since many of the signalling pathways which regulate the expression of CYP24 are also activated by chemotherapeutic drugs, we hypothesised that the drugs could cause the degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 by increasing CYP24 expression, the principal means of facilitating the bio - inactivation and degradation of plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3. Using the kidney cell - lines, COS - 1 and HEK293T cells, we now report that chemotherapeutic drugs, represented by daunorubicin hydrochloride ( an anthracycline antibiotics ), etoposide and vincristine sulphate ( vinca alkaloids and related compounds ) and cisplatin ( an alkylating agent ), were able to enhance CYP24 promoter activity in kidney cell lines transfected with a CYP24 promoter - luciferase construct, either by themselves or in the presencedaunorubicin hydrochloride and etoposide, two of the strongest inducers of CYP24 promoter activation under our experimental conditions, demonstrate that these drugs acted in a concentration - dependent manner. In addition to stimulating promoter activity on their own, the drugs also amplified the induction of the CYP24 promoter by 1,25 ( OH ) 2D3. Synergistic increases were generally observed when the cells were treated simultaneously with 1,25 ( OH ) 2D3 and a drug. The two kidney cell lines generally responded in a similar manner when challenged with the drugs, either in the presence or absence of 1,25 ( OH ) 2D3. Interestingly, the hydroxylated derivative of daunorubicin hydrochloride, doxorubicin hydrochloride which is also a commonly used chemotherapeutic drug, had no effect of promoter activity. Further studies with daunorubicin hydrochloride demonstrated that the effects of the drug per se were not mediated by oxidative stress and the vitamin D receptor was not required for daunorubicin hydrochloride per se to stimulate CYP24 promoter activity. However, daunorubicin hydrochloride caused a modest increase in the expression of the vitamin D receptor and this could contribute to its synergistic activity with 1,25 ( OH ) 2D3. In the presence of etoposide, there was also a tendency for the kidney cells to express higher levels of the vitamin D receptor. A key role for the extracellular signal - regulated protein kinase ( ERK ) 1, ERK2 and ERK5 mitogen - activated protein ( MAP ) kinases was demonstrated for the inductive action of daunorubicin hydrochloride and etoposide, with CYP24 promoter - specific transcription factors located in the first - 298bp being likely targets of the ERK activity. Studies with a dominant negative mutant of MKK4, one of the two immediate upstream activators of the c - jun N - terminal kinase isoforms, demonstrated that this MAP kinase also played a crucial role in inductive actions of the of 1,25 ( OH ) 2D3. Dose - response studies with drugs. Consistent with their use in anti - cancer therapy, all of the above drugs killed the human promyelocytic HL60 leukaemic cells at very low concentrations but had no effect on the viability of kidney or liver cells, either at concentrations used in our experiments or at higher levels. Our data provide novel biochemical evidence that some of the commonly used chemotherapeutic drugs could cause an increase in the transcriptional activation of the promoter, most likely via the MAP kinases activating the transcription factors which bind to the CYP24 promoter. Such an effect could contribute to the reduction in plasma 25 ( OH ) D3 and 1,25 ( OH ) 2D3 in some of the patients undergoing chemotherapy.
Advisor: Hii, Charles Sung Teck
Ferrante, Antonio
Dissertation Note: Thesis (Ph.D.)--University of Adelaide, School of Paediatrics and Reproductive Health, 2005.
Subject: Cancer Chemotherapy Complications.
Cancer Gene therapy.
Cancer in children.
Drugs Side effects.
Keywords: vitamin D metabolism, cancer, chemotherapy, drug testing
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