Diversity in secreted PLA2-IIA activity among inbred mouse strains that are resistant or susceptible to Apc Min/+ tumorigenesis.
The secreted phospholipase A2 type IIA (Pla2g2a) gene was previously identified as a modifier of intestinal adenoma multiplicity in Apc Min/+ mice. To determine if intestinal secreted phospholipase A2 (sPLA2) activity was also attenuated in susceptible strains, we developed a sensitive assay to directly quantitate sPLA2 activity in the murine intestinal tract utilizing a fluorescent BODIPY-labeled phospholipid substrate. Here, we report assay conditions that distinguish between secreted and cytosolic PLA2 enzyme activities in extracts of intestinal tissue. The small intestine exhibited higher activity levels than the large intestine. Consistent with predictions from the sPLA2-IIA gene sequence in inbred strains, we detected low levels of enzyme activity in inbred strains containing sPLA2-IIA mutations; these strains were also associated with greater numbers of intestinal polyps. Additionally, the assay was able to distinguish differences in levels of sPLA2 activity between neoplasia-resistant strains, which were then shown by sequencing to carry variant wild-type sPLA2-IIA alleles. Immunohistochemical analyses of intestinal tissues were consistent with sPLA2-IIA activity levels. This approach enables further studies of the mechanisms of sPLA2 action influencing the development and tumorigenesis of the small intestine and colon in both mice and humans.
Markova
Marina
M
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Koratkar
Revati A
RA
Silverman
Karen A
KA
Sollars
Vincent E
VE
MacPhee-Pellini
Melina
M
Walters
Rhonda
R
Palazzo
Juan P
JP
Buchberg
Arthur M
AM
Siracusa
Linda D
LD
Farber
Steven A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001896:Boron Compounds; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D017491:Genes, APC; D054501:Group II Phospholipases A2; D007150:Immunohistochemistry; D007414:Intestinal Neoplasms / Q000201:enzymology; D007420:Intestine, Large / Q000201:enzymology; D007421:Intestine, Small / Q000201:enzymology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C516288:Pla2g2a protein, mouse