Jan Hein van Dierendonck

In Situ detection of apoptosis during embryogenesis with Annexin V: From whole mount to ultrastructure

Apoptosis is of paramount importance during embryonic development. This insight stems from early studies which correlated cell death to normal developmental processes and now has been confirmed by linking aberrant cell death patterns to aberrant development. Linking apoptosis to the phenotype of a developing organism requires spatial information on the localization of the dying cells, making in situ detection essential This prerequisite limits the tools available for such studies (1) to vital dyes, which can be detected at the whole mount level only; (2) to detection based upon apoptotic morphology by routine light microscopy and electron microscopy; and (3) to staining for apoptosis associated DNA fragmentation via, e.g., the TUNEL procedure, which marks cells in a relative late phase of apoptosis. New apoptosis markers need to be specific and should preferably detect cells early during this process. In the present study we show that the recently discovered in vitro marker of apoptosis, Annexin V meets these requirements for in vivo detection. Through intracardiac injections of biotin labeled Annexin V, a Ca2+ dependent phosphatidylserine binding protein, we were able to visualize apoptotic cells derived from each germ layer in the developing mouse embryo from the whole mount level up to the ultrastructural level. Double-labeling on paraffin sections for both this method and TUNEL revealed that cells become Annexin V-biotin labeled early during the process of apoptosis.

Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.

Development of resistance to glutathione depletion-induced cell death in CC531 colon carcinoma cells: association with increased expression of bcl-2.

The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.

Surgically induced cytokinetic responses in experimental rat mammary tumor models.

The effect of surgical removal of “primary” tumors on the cytokinetics of local tumor remnants, secondary implants, and metastases was investigated in three different rat tumor models in the Wag/Rij rat: a slow‐growing (MCR83) and a fast‐growing (EMR86) hormone‐dependent mammary tumor and a rapidly, but autonomously growing carcinoma (MCR86). The latter two tumors had metastatic potential. Cell kinetic studies were done using in vivo labeling with 5′ ‐ bromodeoxyuridine (BrdUrd). Thirty‐three hours after removal of a subcutaneous MCR83 flank tumor, secondary implants showed a significant (P < 0.05) but transient increase in the BrdUrd labeling index (LI). A more rapid and prolonged increase, lasting for at least 7 days, was observed in EMR86 lymph node and lung metastases. In both models, no effect was observed after sham surgery (consisting of opening and closing of the skin under anesthesia). Removal of MCR86 tumors (growing in the hind leg muscle) also resulted in a rapid, transient LI increase in metastases. Continuous BrdUrd labeling experiments in this tumor model did not favor the hypothesis that the LI increase predominantly resulted from an increase in the growth fraction. Moreover, in this model, the effect was related to operation trauma. A similar increase in LI, although smaller than after tumor removal, was seen after major surgical trauma in MCR83 flank tumors. These results indicate that in the rat, tumor removal and/or major surgical trauma may modulate the cytokinetics of distant metastases significantly. A study of the systemic, possibly endocrine, factors involved in the growth‐stimulating effect of surgical trauma in these rat tumor models may help to assess the clinical relevance of these findings for patients with breast cancer.

Bcl-2 Overexpression Does Not Prevent but Retards Adriamycin Toxicity in CC531 Colon Carcinoma Cells

Background: The Bcl-2 protein is a critical regulator of susceptibility towards cell death induced by antineoplastic drugs. Reduced growth activity and increased glutathione (GSH) levels protect against adriamycin toxicity. We recently demonstrated statistically significantly reduced growth activity and elevated cellular GSH levels in exponentially growing rat CC531 colon carcinoma cells overexpressing the full-length human Bcl-2 protein (CCbcl2#A3). Methods: To assess the importance of reduced growth activity or increased GSH levels, we determined the mitochondrial function, 24 h after adriamycin treatment, in CCbcl2#A3 cells, parental CC531 cells and cells overexpressing the Bcl-2 protein lacking the N-terminal BH4 domain (CCΔBH4): these latter cells contained elevated cellular GSH levels but were not reduced in growth activity. Results: CCbcl2#A3, but not CCΔBH4, cells were 3-fold less susceptible than parental cells suggestive of a protective role for reduced growth but not for increased GSH levels in bcl-2 transfectants. This was confirmed in several growth-inhibited CC531 transfectants and in slowly proliferating (ca. 100% confluent) cell populations compared to exponentially growing (ca. 50% confluent) cell populations. Reduced growth activity might delay the onset of cell death. Therefore, we tested the effect of adriamycin five days after treatment. In this long-term assay we found no differences between the various cells. Conclusion: Reduction of growth activity, for instance by an overexpression of the Bcl-2 protein, only transiently reduced the susceptibility towards adriamycin treatment.

Potentiation of the cytostatic effect of melphalan on colorectal cancer hepatic metastases by infusion of buthionine sulfoximine (BSO) in the rat Enhanced tumor glutathione depletion by infusion of BSO in the hepatic artery

Purpose: Glutathione (GSH) plays an important role in the resistance of tumors to cytostatics. Therefore, depletion of GSH by the GSH synthesis inhibitor buthionine sulfoximine (BSO) has been proposed to enhance the efficacy of certain anticancer agents. We studied the effect of BSO in rats bearing intrahepatically implanted tumors of the CC531 colorectal cancer cell line on the antitumor activity of melphalan (L-PAM). Since these liver tumors tend to derive most of their blood supply from the hepatic artery, we evaluated whether delivery of BSO into the hepatic artery would more selectively decrease GSH levels in the implanted tumor tissue as compared with normal liver and extrahepatic tissues. Methods: Tumor-bearing rats were treated with a 24-h continuous infusion of 0.375 mmol/kg BSO via the jugular vein, immediately followed by a bolus L-PAM (15 μmol/kg; 4.5 mg/kg) infusion via the hepatic artery. Laparotomy was performed on day 14 and 28 after treatment for measurement of the liver tumors. For the evaluation of locoregional administration of BSO, a 24-h continuous infusion of 0.375 mmol/kg BSO was delivered into either the hepatic artery, the portal vein, or the jugular vein in freely moving rats and GSH levels in the tumor, liver, kidney, lung, heart, bone marrow, and blood were measured. Results: BSO infusion via the jugular vein increased the antitumor efficacy of L-PAM injected into the hepatic artery 2-fold as determined at 14 days after treatment. Although infusion of BSO via the hepatic artery depleted GSH more severely in the tumor as compared with jugular vein or portal vein administration, the additional effect was only slight (10%). No difference was observed in any other tissue. Conclusion: GSH depletion increased the cytostatic efficacy of L-PAM 2-fold in vivo as determined at 14 days after treatment. Hepatic artery infusion of BSO translated into a statistically significant, but probably not therapeutically relevant, increase in tumor GSH depletion as compared with the other routes of BSO administration.