# Presentation: 206893

## WHAT IS THE ROLE OF EXTRARENAL VITAMIN D HYDROXYLASE EXPRESSION AND ACTIVITY IN NORMAL AND MALIGNANT CELLS ? 

**HOW IS THIS MODIFIED BY EPIGENETIC MECHANISMS ?**
**HEIDE S. CROSS, MEDICAL UNIVERSITY OF VIENNA, AUSTRIA**

## Enhanced CRC and other cancers have been asso-ciated with low 25-OHD3 levels and not with low 1,25-(OH)2D3, though the latter is the antimitotic agent, however at nM concentrations. 

- Enhanced CRC and other cancers have been asso-ciated with low 25-OHD3 levels and not with low 1,25-(OH)2D3, though the latter is the antimitotic agent, however at nM concentrations.

## VDR AND CYP27B1 EXPRESSION IN HUMAN CRC TISSUE

## METABOLISM OF 25-OH-D3 IN HIGHLY DIFFERENTIATED CACO-2 CELLS

**METABOLISM OF 25-OH-D3 IN HIGHLY DIFFERENTIATED CACO-2 CELLS**

- CYP24-Metabolites

- 1,25-(OH)2-D3

- Late catabolites

**1,25-(OH)****2****-D****3**

**CYP24-Metabolites**

**Late catabolites**

## EXPRESSION OF CYP27B1 PROTEIN IN HUMAN COLON TISSUE

**Immunoblot with an antibody against CYP27B1. Protein was derived from normal colon (NM) and from tumor (G2 and G3) patients (T=tumor; AM=tumor-adjacent mucosa from the same patient)**

**EXPRESSION OF CYP27B1 PROTEIN IN HUMAN COLON TISSUE**

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## HUMAN COLON ADENOCARCINOMA-DERIVED CACO-2 CELLS RESPOND TO AND SYNTHESIZE 1,25-(OH)2D3

**1,25-(OH)****2****D****3 ****(10 nM)**

**25-(OH)D****3**

**1,25-(OH)****2****D****3**

**CYP27B1**

- Cross et al, J Natl Cancer Inst 1992
- Cross et al, J Steroid Biochem Mol Biol 1997

- HUMAN COLON ADENOCARCINOMA-DERIVED CACO-2 CELLS RESPOND TO AND SYNTHESIZE 1,25-(OH)2D3

## IMMUNOFLUORESCENCE POSITIVITY OF

**IMMUNOFLUORESCENCE POSITIVITY OF**

**CYP27B1**

**VDR**

**VDR**

**IN SERIAL SECTIONS OF **
**A and B: ADENOMA**
**C: G3 COLON ADENOCARCINOMA**

**CROSS et al, J Steroid Biochem Mol Biol 97, 121, 2005**

## CYP27B1 AND CYP24 mRNA EXPRESSION IN HUMAN CRC PATIENTS

- Cross et al, Steroid Biochem Mol Biol 2005

## 25-OH-D3 METABOLISM IN G3 COLON TUMOR-DERIVED COGA-13 PRIMARY CULTURES

**25-OH-D3 METABOLISM IN G3 COLON TUMOR-DERIVED COGA-13 PRIMARY CULTURES**

## THE VITAMIN D SYSTEM IN COLON, PROSTATE AND MAMMARY CELL LINES

**THE VITAMIN D SYSTEM IN COLON, PROSTATE AND MAMMARY CELL LINES**

-  **sc   c  +1,25**

**CYP27B1 mRNA**

**CYP24 mRNA**

**VDR mRNA**

## Colon cancer cells: 

**Colon cancer cells: **

**untreated **

**+ 1,25-(OH)****2****-D****3**** **

**Lechner et al, Mol Cell Endocrinol  263, 55, 2007**

**ACTIVITY OF VITAMIN D HYDROXYLASES DETERMINED BY HPLC**

## Prostate cancer cells: 

**Prostate cancer cells: **

**untreated **

**+ 1,25-(OH)****2****-D****3**** **

**Breast cancer cells: **

- untreated

- + 1,25-(OH)2-D3

## REGULATION OF RENAL AND EXTRARENAL VITAMIN D HYDROXYLASES

**  **

**Extrarenal hydroxylase**** expression**** ****is ****regulated in tumor tissue**** during progression****. We provide evidence that vitamin D hydroxylases are regulated by epigenetic events****.**** **

## METHYLATION OF A DNA BASE BY A METHYLTRANSFERASE (Mtase)

**METHYLATION OF A DNA BASE BY A METHYLTRANSFERASE (Mtase)**

**Methylation is an enzyme-mediated chemical modification that adds a methyl group to a selected site. In human DNA, only the cytosine base followed by guanosine (CpG) is affected. Adenosyl Methionine (SAM) is the methyl donor.**

## Hypomethylation is an early event in ageing and cancer and may contribute to chromosomal instability. CAUSES ARE UNKNOWN !  (polymorphisms in MT, lack of availability of methyl groups ?)

**Hypomethylation is an early event in ageing and cancer and may contribute to chromosomal instability. ****CAUSES ARE UNKNOWN !  ****(polymorphisms in MT, lack ****of availability ****of methyl groups ?)**

**HYPERMETHYLATION**
**CpG islands are normally devoid of methylation. Exceptions :  1) genes on the inactive X-chromosome; 2) imprinted genes. **
**When CpG islands in gene promoters are methylated, there is transcriptional repression of the gene. Promoter methylation of certain genes was associated with ageing  and with ****CANCER ****(SUPPRESSOR GENES).**

- ABERRANT METHYLATION CAN BE THE RESULT OF CHANGES IN DNA STRUCTURE, CARCINOGEN EXPOSURE (e.g. MT overexpression), MICROSATELLITE INSTABILITY.

## METHODS TO STUDY CpG ISLAND METHYLATION:

**Distinguish methylated from unmethylated DNA based on methylation-sensitive restriction enzymes that do not cut at methylated restriction sites.**

**Bisulfite sequencing (bisulfite converts unmethylated C to U which is then converted to T during DNA synthesis, but leaves methylated C as is).**

**Methylation Sensitive PCR (MSP): Primers are designed for a certain promoter region, that is either methylated or unmethylated by 5-aza treatment (inhibitor of MT) and this is followed by PCR.**