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ACTIVATION OF APOPTOSIS IN PROM: CASPASE INDEPENDENT?
Ramkumar Menon, M.S., Salvatore J. Lombardi, M.D., Stephen J. Fortunato, M.D.
ABSTRACT
OBJECTIVE: Our laboratory has recently demonstrated an association between apoptosis as a possible inducer of MMP activity. Multiple pathways can trigger apoptosis, the most common one being the caspase cascade. This study examines the mechanism of apoptosis induction in PROM.
METHODS: human fetal membranes were collected from women with PROM, PTL and after term vaginal delivery (n=10). Fragmentation of DNA was studied by LM-PCR (ligase mediated PCR). RNA extracted from fetal membranes was reverse transcribed and a multiple PCR (MPCR) strategy was used to study the expression of caspases 3, 5, 8, 9, and Apaf (caspase activation factor). Quantitative PCR was employed to document the changes in the expression pattern of Bcl-2 and bax.
RESULTS: LM-PCR data showed a ‘DNA ladder’, effect which is typical of DNA fragmentation in human fetal membranes from PROM (8/10) compared to term (3/10) and PTL (0/10). Caspase 3, 9 and 5 were seen in all the samples tested while Apaf and caspase 8 was not detected in any of the samples tested regardless of the clinical condition. A 100 fold increase in bax expression coincided with a significant drop in Bcl-2 expression in PROM membranes compared to the other two groups (p = 0.01). A similar increase in the bax inducer p53 was also seen in PROM.
CONCLUSION: DNA fragmentation was present in fetal membranes from PROM but can be detected only by highly sensitive LM-PCR. Although caspase 3, 5 and 9 genes are expressed, their activator protein genes (Apaf and caspase 8) are not expressed in fetal membranes. DNA fragmentation induced a p53 and bax increase and a drop in the Bcl-2 suggesting a p53 mediated programmed cell death during PROM. These preliminary data indicate the existence of a caspase independent apoptotic pathway in fetal membranes during PROM distinct from the Fas-FasL pathway seen in most of the tissues.
INTRODUCTION
The pathobiology of premature rupture of human fetal membranes (PROM) is still unknown. Multiple factors and pathways that can degrade the fetal membrane extracellular matrix (ECM) region have been proposed. [1] A collagen rich ECM region connects amnion and chorion cells and provides the functional integrity of the membranes throughout pregnancy. [2] A balance between degradation and remodeling activity of the matrix components maintains the integrity of the membranes as they accommodate to the increased volume and pressure during fetal growth. [3] Specific enzymes (matrix metalloproteinases [MMPs]) whose functions are controlled and regulated by tissue specific MMP inhibitors (TIMPs) coordinate these biophysical activities. In pathologic conditions increased endogenous activity of these MMPs is suspected to cause increased ECM degradation and weakening of the membranes leading to rupture. [4]
We have earlier documented that PROM (in vivo) and infection (in vitro) are associated with a molecular imbalance in the ratio between MMPs and TIMPs in human fetal membranes .[5, 6] This increased bioavailability of MMPs may cause membrane degradation and rupture. Significantly some of the MMPs (MMP2/gelatinase A) involved in physiologic matrix turn over and tissue remodeling during normal pregnancy were also shown to increase during PROM. We have recently reported that during PROM MMP2 expression increases and was associated with an increase in the expression of apoptosis (programmed cell death) inducing factors like p53. P53 can transactivate MMP2 gene expression through the AP-2 site in its promoter region. (See figure 1). [7].
Several authors have reported histologic evidence of apoptosis and increase in apoptosis related factors during PROM. However, the studies have not determined the apoptotic pathway in fetal membranes. In most tissues this proceeds through activation of specific cytoplasmic proteases known as caspases (cystine proteases with aspartic acid substrate specificity). Caspase activation leads to morphological and physiological changes resulting in cell death. Two major pathways that lead to the caspase activation have been identified. One pathway involves the family of death receptors at the cell surface (TNF-a-TNFR, Fas-FasL and their related death domains) which directly activates caspases. The second pathway is initiated by damage (fragmentation) to the DNA, which induces proapoptotic proteins like p53 and bax in the cell whose action relocates cytochrome C from mitochondrial membranes to the cytosol. This cytochrome C in turn can activate caspases.
In this report we examined the expression pattern of the caspase family of genes during PROM. We also examined the extent of DNA damage and quantitate the mRNA for proapoptotic elements in the fetal membranes during PROM, preterm labor (PTL) and at term.
MATERIALS AND METHODS
Collection and processing of tissues: Amniochorionic membranes were collected from the following groups of women; Group 1. Women with PROM after cesarean section (four in labor) Group 2. Women with preterm labor (PTL) with intact membranes delivered by cesarean section. Group 3. Term labor with vaginal delivery after uncomplicated pregnancy. Amniochorionic membranes were cut from the placenta, cleansed in Hank’s balanced salt solution for three times to remove adhering blood clots. Decidua was scrapped off with sterile cotton gauze. The membranes were frozen immediately in liquid nitrogen and stored at –70oC until ready for nucleic acid analysis.
Multiple polymerase chain reaction (MPCR) – Total RNA was extracted from tissue using Trizol and 0.5mgm of this RNA was subjected to oligo dT primed reverse transcriptase reaction and cDNA synthesis. A known amount of this cDNA was used for MPCR in which several targets are amplified simultaneously in the same reaction tube (BioSource international, Camarillo, CA) using a Perkin-Elmer thermocycler. In this assay we have used MPCR primers specific for the following mRNAs listed in Table with the expected fragment size.
GAPDH was used as a house-keeping gene with the expected fragment size of 615 bp. The following parameters were used for PCR; initial denaturation at 95o C for 1 min followed by 2 cycles at 95 oC for 1 min and 56 oC for 4 min. Another 33 cycles were run at a denaturation temperature of 94 oC for 1 min and 56 oC for 2.5 min. The PCR products were analyzed on a 1.2% ethidium bromide stained agarose gel with 100 bp DNA ladder as molecular weight marker. A cDNA sample from cells undergoing apoptosis, which expressed all the genes under investigation was used as positive control. (Figure 2a) A sample of RNA subjected to the RT reaction without the enzyme was used as the negative control.
LM-PCR and Quantitative PCR (QPCR)- Ligase mediated PCR was performed to check the extent of DNA damage in fetal membranes. QPCR was performed to quantitate the mRNA for p53, bax (proapoptotic) and Bcl-=2 (anti-apoptotic). The detailed protocols are listed in our earlier publications.
|
Gene of Interest |
Fragment Size |
|
Apaf (caspase activator) |
498 bp |
|
Caspase 3 |
318 bp |
|
Caspase 5 |
256 bp |
|
Caspase 8 (FLICE) |
405 bp |
|
Caspase 9 |
200 bp |
Figure 2a. Control experiment: mRNA extracted from cells undergoing apoptosis which express all genes under investigation. GAPDH was used as a ‘house keeping gene’. MPCR was performed only after documenting GAPDH expression in each samples.
2. mRNA expression for caspase 8 (FLICE) and apoptosis activation factor (Apaf-1- caspase activation factor) was never seen in fetal membranes. (Figure 2b)
3. DNA fragmentation as evidenced by classical DNA laddering was seen in 80% of PROM membranes and 25% of the PTL membranes. DNA smearing was seen in a majority of term membranes indicating 	necrosis instead of apoptosis. 30% of term membranes showed apoptosis. (Figure 3)
Proapoptotic antigens like p53 and bax were significantly elevated in PROM compared to term and PTL groups. Elevation
of antiapoptotic Bcl-2 expression was seen in PTL. Term membranes did not show Bcl-2. (Figure 4)
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CONCLUSIONS
- Expression of caspase 3, 5 and 9 and the total absence of mRNA for Apaf-1 (caspase
activator) and caspase 8 mRNAs suggests that caspase mediated programmed cell death is unlikely in fetal membranes.
- PROM membranes undergo some DNA fragmentation which can be documented only by highly
sensitive techniques like LM-PCR.
- Apoptosis during PROM is likely initiated by DNA fragmentation and fragmentation induced p53 increasep53 mediated bax increase and Bcl-2 suppression.
DISCUSSION
- Apoptosis appears to be involved in PROM.
- Apoptosis proceeds through the p53 mediated caspase independent pathway.
- p53 likely promotes MMP activation leading to membrane degradation and rupture.
REFERENCES
- French JI, McGregor JA. The pathobiology of premature rupture of membranes. Semin Perinatol 1996; 20:344-368
- Kanayama N, Terao T, Kawashima Y et al. Collagen types in normal and prematurely ruptured amniotic membranes. Am J Obstet Gynecol 1985; 153:899-903.
- Vadillo-Ortega F, Gonzalez-Avila G, Selman M et al. Collagen metabolism in premature rupture of membranes. Obstet Gynecol 1990; 75:84-88.
- Vadillo-Ortega F, Gonzalez-Avila G, Furth EE et al. 92 kd Type IV collagenase (matrix metalloproteinase-9) activity in human amnionchorion increases with labor. Am J Pathol 1995; 146:148-156.
- Fortunato SJ, Menon R, Lombardi SJ. MMP/TIMP imbalance in amniotic fluid during PROM: An indirect support for endogenous pathway to membrane rupture. J Perinat Med, 1999; 27:362-368.
- Fortunato SJ, Menon R, Lombardi SJ. Amniochorion gelatinase/gelatinase inhibitor imbalance in vitro: A possible infectious pathway to rupture. Obstet Gynecol 2000, 240—244.
- Fortunato SJ, Menon R, Lombardi SJ. Programmed cell death (apoptosis): A possible
pathway to metalloproteinase activation and fetal membrane degradation in PROM. Am J Obstet, In Press.