The effects of radiation on vitamin D actions in normal mammary cells, by Natalia L. Martinez

1. The Role of Vitamin D in Breast Cancer :
The effects of radiation on vitamin D actions in
normal mammary cells
Department of Chemistry, The College of New Rochelle
Natalia L. Martinez
Mentor: Elvira Longordo
University at Albany Summer Research Program
GEN*NY*Sis Center for Excellence in Cancer Genomics
JoEllen Welsh and Erika LaPorta

2. Breast Cancer
• Breast Cancer is the uncontrolled growth of
breast cells, in the lobules or the ducts
Normal Cancer

3. Cellular Origin of Cancer

4. What Causes Cancer?
Intrinsic Factors
• Heredity
• Diet
• Hormones
Extrinsic Factors
• Chemicals
• Radiation

5. DNA Damage
• Chemicals and Radiation can
damage DNA
• Cells can sense and repair
damage
– p53- acts in the damage response
mechanism
– tumor suppressor
• There is now a focus on
identifying additional agents
that enhance DNA repair
•http://www.haemophilia.org.nz/DBimages/FCK/Image/dna.jpg

6. The link between Cancer &
vitamin D
• Recent research has shown a link
between vitamin D and the prevention
and treatment of Breast Cancer
• Role of Vitamin D
–Regulates Calcium and Phosphorus levels in
the blood
–Prevention of Rickets

7. How is Vitamin D obtained?
• Food Sources
– Fatty Fish
– Eggs
– Fortified Foods
• Sunlight Exposure
– UVB light

8. From Vitamin D to 1α,25(OH)2D3
7-dehydrocholesterol
found in the skin
cholecalciferol
Or
vitamin D3.
forming
25- hydroxycholecalciferolProducing the active form
of vitamin D3,
1,25-dihydroxycholecalciferol.

9. UV Stimulated Synthesis of Calcitriol
Biochemistry Fifth Ed. Berg, Tymoczo and Stryer. W.H. Freeman, 2002.

10. The vitamin D receptor (VDR)
• It is found in the nucleus of different tissues
through the body
• VDR is a nuclear hormone receptor
• VDR is the major mediator of 1,25D actions

11. 1,25D & VDR
When the active
form of vitamin D
binds to its nuclear
receptor VDR it
regulates gene
expression which
influences cellular
proliferation,
differentiation and
apoptosis

12. In My Project:
• Study the effects of radiation on vitamin D
actions in normal human mammary cells (HME)
Hypothesis:
• DNA damage caused by ionizing radiation will up-
regulate p53 and VDR expression
Western Blot
• Protection of the cells can be achieved by the
ligand binding of 1,25D3 to VDR in damaged cells
Crystal violet assay & immunoflurescent microscopy

13. Investigated the effects of
radiation on vitamin D receptor
(VDR) expression

14. Procedure
• Cell Culture
-HME cells (9 plates)
• Ionizing Radiation
DOSES OF EXPOSURE Incubation Period.
Sham-0KV-0mA; 1min. 3 plates –analyzed after 1hr.
2Gy- 130KV- 2mA; 1min. 3 plates- analyzed after 3hr.
8Gy- 130KV- 7.7mA; 1 min 3 plates- analyzed after 6hr.
http://www.instapstudycenter.net/images/faxitron.GIF

15. Detection of p53 and VDR by
Western Blot
Lamin A/C
Lamin A/C
8Gy
2Gy
Sham.
8Gy.
2Gy
Sham
Sham
2Gy
8Gy
p53
VDR
6hrs. 3hrs. 1hr.

16. Use immunofluorescence microscopy to monitor
expression and sub-cellular localization of VDR after
radiation exposure in the presence and absence of 1,25D
Detection of VDR by
Confocal Microscopy
www.ion.ucl.ac.uk/.../neuropath/techniques.htm

17. Results
1,25DEtOH

18. Results – 1hr

19. Results – 3hr

20. Examine whether treatment
with 1,25D alters HME cell
response to radiation.

21. Procedure
• Plated cells in 6 well plates
• Treated with 100nM Vitamin D
– 48hr. Prior to radiation exposure
– Just before radiation exposure
• Cultured for 4 days
• Quantified cell density using Crystal Violet Assay

22. Morphology
Sham-EtOH
Sham-VitD
2Gy-EtOH
2Gy-VitD

23. Results
Pretreated
EtOH Sham EtOH 2Gy VitD Sham VitD 2Gy
0
1
2
3
* *
Treatment
Absorbance590
Cotreated
EtOH Sham EtOH 2Gy VitD Sham VitD 2Gy
0
1
2
3
* *
Treatment
Absorbance590

24. Conclusion
• HME cells express VDR & p53 proteins
• Radiation may increase VDR & p53 expression
• VDR localization is altered by radiation treatment
• Vitamin D inhibits growth of HME cells

25. Acknowledgments
• Dr. JoEllen Welsh
• Erika LaPorta
• Dr. Meggan Keith
• Dr. Judy Narvaez
• Dr. Christopher Fernando
• Deanna Morehead
• UASRP Staff and Scholars