Edinboro University of Pennsylvania

1
Detecting
Daunomycin
Induced
Intrachromosomal
Recombination in
Saccharomyces cerevisiae
using a Del Assay
Jafa Armagost
Alexis Snow
Laura Egli
William J. Mackay

• Edinboro University of Pennsylvania

2
Daunomycin

• Anthracycline
• Antitumor antibiotic
• Streptomyces sp.
• Hematological and Myelogenous cancers

3
Dauomycin
Suarato, A. et al. 1995
4
DNA-Daunomycin Complex
Inhibits DNA replication (Antitumor effect)
Intercalation Interstrand crosslinks
5
Previous Research
Dr. Koch, Colorado University at Boulder
G
G
DNA-DNA Interstrand Crosslink
Friedberg et al. (1995) Taatjes et al. (1997)
6
Side Effects

• Cytotoxic effects
• Cardiotoxicity
• Acute Myelosuppression
• Secondary Cancers

7
Previous Research

• Ames Assay
• Prokaryotic mutation test
• Daunomycin is mutagenic

Mackay et. al 2000 Texas Journal of Science 52
(3) 223-229 (August 2000)/ 53(3) 239-246
(August 2001) / 54 (3) 249-260 (August 2002)
8
Saccharomyces cerevisiae

• Eukaryotic Unicellular Organism
• Extensive genetic history
• Genome Sequenced

9
Del Assay

• Developed by Dr. Robert Schiestl (1989)
• Eukaryotic recombination assay
• Detects mutagenic/non-mutagenic carcinogens

Schiestl RH, Gietz RD, Mehta RD, and Hastings PJ
Carcinogenesis 1989 10(8)1445-1455
10
Saccharomyces cerevisiae Strain RS112

• Mata/a
• leu2-3,112/leu2-?98
• ade2-40/ade2-101
• HIS3 pRS67/his3-?200 (his33'- LEU2-
  pBR322-his35')

Schiestl RH, Gietz RD, Mehta RD, and Hastings PJ.
Carcinogenesis 1989 10(8)1445-1455
11
Intrachromosomal Recombination
( HIS3 )
12
( HIS3
)
HIS3
HIS3
13
HIS3
pBR322
pRS67
his3
LEU2
14
Media
SC-LEU (Synthetic complete LEU) selects for
Leu prototrophs
SC-HIS (Synthetic complete HIS) selects for
His prototrophs

• YPAD (Yeast Peptone Dextrose ADE)
• Background

15
Chemicals

• Daunomycin
• 170 mg/ml
• 340 mg/ml
• Positive Control
• Methyl Methanesulfonate (MMS)
• 65 mg/ml
• 130 mg/ml

Schiestl RH, Gietz RD, Mehta RD, and Hastings PJ
Carcinogenesis 1989 10(8)1445-1455
16
Procedure for Del Assay
17
HIS3
HIS3
2. Culture was exposed to daunomycin or MMS and
grown at 30o C for 17 hr
18
3. Exposed culture was plated on
SC-HIS
HIS3
pBR322
pRS67
his3
LEU2
4. Incubated for 2 days at 30o C
5. Colonies were counted
19
Mutation Frequency
Mutants
Mutation Frequency
Population
20
Induction Fold
Induced Mutation Frequency
Induction Fold
Spontaneous Mutation Frequency
21
Results of Del Assay
22
Conclusion

• Daunomycin has tested positive in a
• Yeast Del assay making it a possible
• CARCINOGEN

23
Future Research I

• Del Assay
• Daunomycin
• Idarubicin
• Adriamycin
• Epirubicin

OH
O
CCH3
OH
OCH3
O
OH
OCH3
O
O
H3C
HO
NH2 HCl
24
Future Research I
Del Assay Daunomycin Idarubicin
Adriamycin Epirubicin
OH
O
CCH3
OH
O
OH
O
O
H3C
HO
NH2 HCl
25
Future Research I
Del Assay Daunomycin Idarubicin
Adriamycin Epirubicin
OH
O
CCH2OH
OH
OCH3
O
OH
OCH3
O
O
H3C
HO
NH2 HCl
26
Future Research I
Del Assay Daunomycin Idarubicin
Adriamycin Epirubicin
OH
O
CCH2OH
OH
OCH3
O
OH
OCH3
O
O
H3C
HO
NH2 HCl
27
Future Research II

• Mouse in vitro Del Assay
• p gene
• pun

Schiestl RH, Aubrecht J, Khogali F, Carls N.
Proc. Natl. Acad. Sci. 1997 94 (9) 45764581
28
Future Research II
Inject pun fetuses with daunomycin
Check mice for induced spotting
Schiestl RH, Aubrecht J, Khogali F, Carls N.
Proc. Natl. Acad. Sci. 1997 94 (9) 45764581
29
Acknowledgements

• Dr. Chris Sommers, U.S.D.A.

30
Grants

• Pennsylvania Academy of Science
• Tri-Beta Biological Honor Society

31
Previous Students

• Anthony Cauchi (Univ. of Michigan)
• Ames assay with Daunomycin
• Lisa Phelps (LECOM)
• Ames assay with Adriamycin

32
Previous Students

• John Brumfield (LECOM)
• Ames assays with Epirubicin and Idarubicin
• Natalya Teygart (LECOM)
• Proteins that repair Dauomycin induced DNA
  adducts