Longitudinal mechanical tension induces growth in the small bowel of juvenile rats.

INTRODUCTION: The aim of our study was to apply longitudinal force to the small bowel to increase the length of intestine in juvenile rats. METHODS: Fifty juvenile rats had double barrelled, blind loop ostomies created using an isolated segment of bowel. Our intestinal lengthening device was inserted into one of the loops and the second loop served as a control. Once the device was deployed, the experimental, control, and in situ segments of bowel were evaluated for length, weight, histology, and disaccharidase enzyme activity. RESULTS: Mechanical tension increased intestinal length by 149%. The lengthened bowel also exhibited a greater total weight (218%), greater mucosal weight (122%), and increased protein mass (164%) compared with the control limb of bowel. Histologically, there was a markedly increased thickness of the muscularis propria in the lengthened bowel (200% increase compared with the control limb). Functionally, we found increased total disaccharidase activity in the lengthened bowel (between 47% and 350%, depending on the particular enzyme tested; p<0.01). CONCLUSION: Mechanical tension induces intestinal growth by increasing length, weight of the bowel and mucosa, and protein mass. Histological changes, such as increases in Paneth cells, suggest that increased proliferation and reorganisation of the mucosa and muscularis propria are a response to mechanical tension. Functionally, increased intestinal length corresponds with increased disaccharidase activity, thus implying potential increased absorptive capacity of the lengthened bowel.

A redox-inactive thioredoxin reduces growth and enhances apoptosis in WEHI7.2 cells.

Cancer cell lines transfected with thioredoxin show increased anchorage-independent growth and decreased sensitivity to induction of apoptosis by a number of anticancer drugs. The present studies were undertaken to evaluate further the role of thioredoxin in cell growth and drug-induced apoptosis. A redox-inactive mutant thioredoxin was stably transfected into WEHI7.2 mouse lymphocytic leukemia cells and two clones were examined for growth characteristics and the induction of apoptosis by dexamethasone, etoposide, doxorubicin, and staurosporine. These clones each exhibited a 71% increase in doubling time in solution and a 20 and 75% reduction in colony formation in soft agarose. The transfected cells also showed increased susceptibility to apoptosis induced by dexamethasone, etoposide, doxorubicin, and staurosporine compared with controls. The results of this study suggest that thioredoxin can regulate the growth rate of cells and that thioredoxin is a critical component in the pathway leading to drug-induced apoptosis in WEHI7.2 cells.

Nuclear factor kappaB transactivation is increased but is not involved in the proliferative effects of thioredoxin overexpression in MCF-7 breast cancer cells.

Thioredoxin (Trx) is a small redox-active protein that provides reducing equivalents for key cysteine residues of proteins through thiol-disulfide exchange, such as the transcription factor nuclear factor-kappaB (NF-kappaB). NF-kappaB activation has been associated previously with cell growth and the inhibition of apoptosis. We have shown in earlier studies that overexpression of Trx in MCF-7 cells increases anchorage-independent growth. In this study, the activation of NF-kappaB was examined as a mechanism through which Trx overexpression might promote anchorage-independent growth. Constitutive NF-kappaB activity is elevated 4-7-fold in Trx-overexpressing cells. NF-kappaB activity was inhibited in these cells by expressing a dominant-negative mutant of the IkappaB alpha protein (IkappaB alphaM). Expression of IkappaB alphaM in Trx-overexpressing cells dramatically reduced the Trx-associated increase in NF-kappaB activity but did not affect anchorage-dependent or -independent growth. The results suggest that increased growth in MCF-7 cells overexpressing Trx is not mediated by increased activation of the transcription factor, NF-kappaB. Additionally, activator protein-1 (AP-1), another transcription factor associated with growth, was increased up to 10-fold in Trx-overexpressing cells. Thus, AP-1 activation might contribute to the growth-promoting effect of Trx.

Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2.

The effects of the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 were examined with respect to modulation of 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis in human myeloid leukemia cells (HL-60) overexpressing the antiapoptotic protein Bcl-2. HL-60/Bcl-2 cells displayed a 5-fold increase in Bcl-2 protein compared with empty-vector counter-parts (HL-60/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-xL. After exposure to an equimolar concentration of ara-C (10 microM for 6 hr), HL-60/Bcl-2 cells were significantly less susceptible to apoptosis, DNA fragmentation, and loss of clonogenicity than HL-60/pCEP4 cells. The protective effect of increased Bcl-2 expression was manifested by a failure of ara-C to induce activation/cleavage of the Yama protease (CPP32; caspase-3) and degradation of one of its substrates, poly(ADP-ribose)polymerase to an 85-kDa cleavage product. When HL-60/Bcl-2 cells were preincubated with bryostatin 1 (10 nM; 24 hr) or coincubated with either staurosporine (50 nM; 6 hr) or UCN-01 (300 nM; 6 hr) after a 1-hr preincubation, exposures that exerted minimal effects alone, ara-C-induced apoptosis and DNA fragmentation were restored to levels equivalent to, or greater than, those observed in empty-vector controls. These events were accompanied by restoration of the ability of ara-C to induce CPP32 cleavage and activation, poly(ADP-ribose) polymerase degradation, and inhibition of colony formation. Western analysis of Bcl-2 protein obtained from overexpressing cells treated with bryostatin 1, staurosporine, or UCN-01 revealed the appearance of a slowly migrating species and a general broadening of the protein band, effects that were insensitive to the protein synthesis inhibitor cycloheximide. Alterations in Bcl-2 protein mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reversed by treatment of lysates with alkaline phosphatase or protein phosphatase 2A; actions of the latter were blocked by the specific phosphatase inhibitor okadaic acid. In vivo labeling studies of Bcl-2 protein demonstrated increased incorporation of [32PO4]orthophosphate in drug-treated cells. Last, phosphorylated Bcl-2 failed to display decreased binding to the proapoptotic protein Bax. Collectively, these findings indicate that bryostatin 1, which down-regulates PKC, and staurosporine and UCN-01, which directly inhibit the enzyme, circumvent resistance of Bcl-2-overexpressing leukemic cells to ara-C-induced apoptosis and activation of the protease cascade. They also raise the possibility that modulation of Bcl-2 phosphorylation status contributes to this effect.

Potentiation of ara-C-induced apoptosis by the protein kinase C activator bryostatin 1 in human leukemia cells (HL-60) involves a process dependent upon c-Myc.

The role of the nuclear phosphoprotein c-Myc has been examined with respect to the regulation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human leukemia cells exposed to bryostatin 1 and other pharmacologic protein kinase C (PKC) activators. Pretreatment of HL-60 cells for 24 hr with 10 nM bryostatin 1 significantly potentiated the ability of ara-C (10 microM; 6 hr) to induce apoptosis without reducing the expression of c-Myc protein. In contrast, equivalent exposure to the stage 2 tumor-promoting PKC activator mezerein (10 nM) in conjunction with ara-C reduced c-Myc levels by 87% and failed to potentiate apoptosis. Co-administration of bryostatin 1 with mezerein before ara-C prevented down-regulation of c-Myc and augmented cell death, whereas co-treatment with the calcium ionophore A23187 (250 nM) and bryostatin 1 reduced c-Myc levels by 80% and abrogated the increase in ara-C-induced apoptosis. When cells were exposed for 24 hr to a c-myc antisense oligonucleotide (AS-ODN;10 microM) but not to a scrambled sequence ODN (SS-ODN) prior to ara-C, c-Myc expression was reduced by 81%, and apoptosis and cell viability were unperturbed. However, AS-ODN (but not SS-ODN) reduced c-Myc protein in cells pre-exposed to bryostatin 1 by 74% and abrogated potentiation of ara-C-induced apoptosis. The actions of c-myc AS-ODN did not stem from proximal G1 arrest/differentiation or biochemical events, since they were not associated with a reduction in the S-phase cell fraction, p21(WAF1/CIP1) induction, pRb hypophosphorylation, or alterations in ara-C metabolism. Together, these findings indicate that HL-60 cell apoptosis proceeds by both c-Myc-dependent and -independent pathways, and that only the former are involved in the potentiation of ara-C-mediated cell death by bryostatin 1.

Modulation of the expression of Bcl-2 and related proteins in human leukemia cells by protein kinase C activators: relationship to effects on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis.

We have previously reported that pretreatment of HL-60 human promyelocytic leukemia cells with the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 potentiates induction of apoptosis by the antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) (Biochem Pharmacol 47:839,1994). To determine whether this phenomenon results from altered expression of Bcl-2 or related proteins, Northern and Western analysis was employed to assess the effects of bryostatin 1 and other PKC activators on steady-state levels of Bcl-2, Bax, Bcl-x, and Mcl-1 mRNA and protein. Pretreatment of cells for 24 h with 10 nM bryostatin 1, or, to a lesser extent, the stage-1 tumor-promoter phorbol dibutyrate (PDB) significantly potentiated apoptosis induced by ara-C (100 microM; 6 h); in contrast, equivalent exposure to the stage-2 tumor promoter, mezerein (MZN), which, unlike bryostatin 1, is a potent inducer of differentiation in this cell line, failed to modify ara-C-related cell death. Neither bryostatin 1 nor PDB altered expression of bcl-2/Bcl-2 over this time frame. In contrast, MZN down-regulated bcl-2 mRNA levels, but this effect was not accompanied by altered expression of Bcl-2 protein. None of the PKC activators modified expression of Bax or Bcl-x(L) mRNA or protein; levels of Bcl-x(S) were undetectable in both treated and untreated cells. However, expression of Mcl-1 mRNA and protein increased modestly after treatment with either bryostatin 1 or PDB, and to a greater extent following exposure to MZN. Combined treatment of cells with bryostatin 1 and MZN resulted in undiminished potentiation of ara-C-mediated apoptosis and by antagonism of cellular maturation. These effects were accompanied by unaltered expression of Bcl-2, Bax, and Bcl-x(L), and by a further increase in Mcl-1 protein levels. When cells were co-incubated with bryostatin 1 and calcium ionophore (A23187), an identical pattern of expression of Bcl-2 family members was observed, despite the loss of bryostatin 1's capacity to potentiate apoptosis, and the restoration of its ability to induce differentiation. Finally, treatment of cells with bryostatin 1+/-ara-C (but not ara-C alone) resulted in a diffuse broadening of the Bcl-2 protein band, whereas exposure of cells to taxol (250 nM, 6 h) led to the appearance of a distinct Bcl-2 species with reduced mobility, phenomena compatible with protein phosphorylation. Together, these findings indicate that the ability of bryostatin 1 to facilitate drug-induced apoptosis in human myeloid leukemia cells involves factors other than quantitative changes in the expression of Bcl-2 family members, and raise the possibility that qualitative alterations in the Bcl-2 protein, such as phosphorylation status, may contribute to this capacity. They also suggest that increased expression of Mcl-1 occurs early in the pre-commitment stage of myeloid cell differentiation, and that this event does not protect cells from drug-induced apoptosis.

Effects of antisense p21 (WAF1/CIP1/MDA6) expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (HL-60).

The p21MDA6 gene product induces cell cycle arrest in p53-null human leukemic cells exposed to differentiation stimuli. We employed an HL-60 cell line stably transfected with a p21MDA6 antisense construct to compare the effects of p21MDA6 dysregulation on the response of myeloid leukemia cells to differentiating and cytotoxic agents. Antisense-expressing cells (HL-60/AS5) treated with 5 nM PMA for 24 h exhibited attenuated induction of p21MDA6 compared to empty vector controls (HL-60/V2). This phenomenon was accompanied by a reduction in the percentage of cells undergoing G1 arrest (67.6 +/- 4.7 vs 82.9 +/- 1.3; P < or = 0.01) and expressing the monocytic maturation marker cd11b (35.5 +/- 2.8 vs 50.5 +/- 2.4; P < or = 0.005). Although HL-AS5 and HL-60/V2 cells did not exhibit obvious differences in the phosphorylation status of the retinoblastoma protein (pRB), in E2F complex formation, or in p27klp1 induction following PMA exposure, inhibition of activity of cyclin-dependent kinase-2 was attenuated in the antisense-expressing line. A 24-h exposure to 5 nM PMA also reduced the cloning efficiency of HL-60/V2 cells to a significantly greater extent than HL-60/AS5 cells (ie to 30.1 +/- 7.0 vs 57.2 +/- 5.6 of controls; P < or = 0.01). In contrast to the disparate responses to PMA, HL-60/AS5 and HL-60/V2 cells treated with the antimetabolite 1-beta-D-arabinofurano-sylcytosine (Ara-C; 10 microM for 6 h) displayed equal susceptibility to G1 arrest, apoptosis, and inhibition of clonogenicity, phenomena unaccompanied by p21MDA6 and p27klp1 induction, or pRB dephosphorylation. These observations indicate that dysregulation of p21MDA6 in p53-null human myeloid leukemia cells interferes with PMA-related G1 arrest, CDK-2 inhibition, differentiation, and loss of clonogenic survival in the absence of obvious alterations in pRB phosphorylation status or E2F complex formation. They also provide functional evidence that p21MDA6 induction does not appear to be required for Ara-C-induced apoptosis, G1 arrest, or the resulting reduction in the self-renewal capacity of HL-60 cells.

Coordinate regulation of stress- and mitogen-activated protein kinases in the apoptotic actions of ceramide and sphingosine.

We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic leukemia cells. Acute exposure of U937 cells to either lipid resulted in loss of proliferative capacity, degradation of genomic DNA, and manifestation of apoptotic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and failed to modify c-jun/c-Jun expression. Dominant-negative blockade of normal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was without effect on those of sphingosine, indicating that ceramide-related apoptosis is directly dependent on activation of c-Jun, whereas sphingosine-induced cell death proceeds via an unrelated downstream mechanism. Characterization of the mitogen-activated protein kinase (MAPK) cascade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/ p44-ERK2 activity was gradually reduced by ceramide but immediately and completely suppressed by sphingosine. Moreover, blockade of the MAPK cascade by the aminomethoxyflavone MEK1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and induced apoptosis in a manner qualitatively resembling that of sphingosine. Both lipids sharply increased p38-RK activity; selective pharmacological inhibition of p38-RK by the pyridinyl imidazole SB-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphingosine, suggesting that p38-RK is not essential for lipid-induced apoptosis. These findings demonstrate that reciprocal alterations in the SAPK and MAPK cascades are associated with the apoptotic influence of either lipid inasmuch as (i) ceramide-mediated lethality is primarily associated with strong stimulation of SAPK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated with weak stimulation of SAPK and strong inhibition of MAPK. We therefore propose that leukemic cell survival depends on the maintenance of an imbalance of the outputs from the MAPK and SAPK systems such that the dominant basal influence of the MAPK cascade allows sustained proliferation, whereas acute redirection of this balance toward the SAPK cascade initiates apoptotic cell death.

Modulation of protein kinase C activity and calcium-sensitive isoform expression in human myeloid leukemia cells by bryostatin 1: relationship to differentiation and ara-C-induced apoptosis.

Previous studies have shown that pretreatment of human myeloid leukemia cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PKC activity and/or Ca2+-dependent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-2 tumor promoter mezerein under conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nM bryostatin 1, which does not induce differentiation in this cell line, led to a profound reduction in membrane and cytosolic PKC activity, decreased expression of cPKC isoforms (alpha, betaI, betaII, gamma), and a marked increase in ara-C induced apoptosis. In contrast, 10 nM mezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as alpha, betaI, and gamma cPKC isoform expression, and failed to potentiate ara-C-related apoptosis. The effects of bryostatin 1 were dominant to those of mezerein, in that the combination resulted in down-regulation of PKC activity and expression and potentiation of ara-C-induced apoptosis, but not cellular maturation. However, coadministration of the Ca2+ ionophore A23187 (250 nM) restored bryostatin 1's differentiating ability while antagonizing its capacity to augment apoptosis, despite failing to reverse bryostatin 1-induced down-regulation of PKC activity and cPKC isoform expression. Furthermore, pretreatment of differentiation-responsive monocytic leukemia cells (U937) with bryostatin 1 substantially reduced PKC activity and cPKC isoform expression, but exerted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1 after ara-C dramatically increased apoptosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regulation of total assayable PKC activity and cPKC expression by bryostatin 1 are insufficient, by themselves, to account for potentiation of leukemic cell apoptosis, at least under conditions in which differentiation occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell differentiation and drug-induced apoptosis.

Effect of AS101 on bryostatin 1-mediated differentiation induction, cell cycle arrest, and modulation of drug-induced apoptosis in human myeloid leukemia cells.

Based upon earlier reports of synergism in cells of lymphoid origin, we have examined interactions between the organotellurium compound AS101 and the protein kinase C (PKC) activator bryostatin 1 with respect to differentiation and Ara-C-induced apoptosis in human myeloid leukemia cells (HL-60). Although preincubation with bryostatin 1 (10 nM) for 24 h significantly increased DNA fragmentation and apoptosis in cells subsequently treated with 10 microM Ara-C for 6 h, this effect was not enhanced by co-administration of AS101 (1.5 microM). However, while exposure of cells to AS101 or bryostatin 1 alone for 72 h was ineffective in inducing cellular maturation, combined treatment resulted in the induction of differentiated features in a subset of cells, manifested by an increase in cell adherence, CD11b expression, cytoplasmic granularity and cell spreading. In addition, cells exposed to the combination of AS101 and bryostatin 1, in contrast to cells incubated with these agents individually, displayed a significant decline in the S-phase and a corresponding increase in the G0/G1 cell populations. These events were accompanied by an increase in protein expression of the cyclin-dependent kinase inhibitor, p21 (WAF1/CIP1/MDA6), and a decline in expression of the c-myc protein. AS101 failed to increase intracellular free Ca2+ ([Ca2+]i) in HL-60 cells, or reverse the profound PKC down-regulation induced by bryostatin 1. Whereas treatment of cells with 1.5 microM AS101 or 10 nM bryostatin 1 for 24 h exerted minimal growth inhibitory effects, combined exposure to these agents reduced colony formation by over 70%. Finally, although addition of AS101 did not potentiate apoptosis induced by the bryostatin 1/Ara-C combination, it did lead to a further reduction in clonogenicity. Together, these findings demonstrate that AS101 partially restores the ability of bryostatin 1 to trigger a differentiation program in an otherwise unresponsive HL-60 cell line, possibly by facilitating bryostatin 1-mediated G1 arrest. They also indicate that AS101 potentiates the antiproliferative effects of bryostatin 1 administered alone or in combination with Ara-C through a mechanism other than, or in addition to, induction of apoptosis.

Effect of 1-beta-D-arabinofuranosylcytosine on apoptosis and differentiation in human monocytic leukemia cells (U937) expressing a c-Jun dominant-negative mutant protein (TAM67).

The proto-oncogene c-jun encodes a component of the AP-1 transcription-activating complex and has been implicated in the regulation of diverse cellular processes, including cell proliferation, differentiation, transformation, and most recently, apoptosis. We have used a U937 monocytic leukemia cell line stably expressing a c-jun dominant-negative, transactivation-domain deletion mutant (TAM67) to assess the role of c-jun in apoptotic events induced by exposure to the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C). Mutant cells produce a truncated M(r) 29,000 protein that interferes with the function of normal c-Jun (and c-Fos) proteins through a quenching mechanism. Parental U937, cells expressing TAM67, and cells carrying only the empty vector (pMM) were exposed to ara-C for 6 h, and apoptosis was monitored by cell morphology as well as qualitative and quantitative assays of DNA damage. No differences in apoptosis could be detected between the three cell lines at any of the ara-C concentrations evaluated. In addition, ara-C concentrations > or = 1.0 x 10(-6) M were equally inhibitory to the clonogenic growth of U937 and TAM67-expressing cells. In contrast, lower concentrations of ara-C (i.e., < 5.0 x 10(-7) M) were significantly less inhibitory to mutant U937 cell colony formation than to their parental counterparts. The reduced sensitivity of TAM67-expressing cells to low concentrations of ara-C could not be attributed to biochemical or cytokinetic factors, since the two cell lines were indistinguishable with respect to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) formation, ara-CTP:dCTP ratios, and S-phase fraction. However, a significantly lower percentage of TAM67-expressing cells exposed to submicromolar concentrations of ara-C exhibited features associated with a differentiated monocytoid phenotype (i.e., increased plastic adherence and CD11b expression) compared to their parental counterparts. Lower concentrations of ara-C were also significantly less effective in decreasing the percentage of S-phase cells and in down-regulating c-myc mRNA levels in the mutant line, events associated with induction of leukemic cell differentiation. Finally, ara-C-induced up-regulation of c-jun message and protein was markedly attenuated in TAM67-expressing cells, findings consistent with a c-jun dominant-negative model. Collectively, these findings suggest that dysregulation of c-jun in U937 cells antagonizes low-dose ara-C-mediated cellular maturation but does not prevent higher concentration of this agent from triggering apoptosis. They also raise the possibility that separate aspects of the antiproliferative actions of ara-C may be differentially regulated by c-jun.

Role of c-jun in human myeloid leukemia cell apoptosis induced by pharmacological inhibitors of protein kinase C.

Recent study results suggest that protein kinase C [PKC (EC 3.1.4.3)] -dependent up-regulation of c-jun may be involved in leukemic cell programmed cell death, or apoptosis, occurring in response to various chemotherapeutic agents. The current study was undertaken to further evaluate the contribution of c-jun in apoptosis with the use of two highly specific pharmacological inhibitors of PKC (calphostin C and chelerythrine). To address this issue, two human leukemic cell lines, HL-60 and U937, and a U937 subline stably expressing a dominant negative c-jun mutant (TAM67) were exposed to calphostin C and chelerythrine, and c-jun expression was monitored at both the mRNA and protein levels. Both PKC inhibitors induced the classic morphological features of apoptosis as well as internucleosomal DNA degradation in a concentration- and schedule-dependent manner. Concomitant with these changes, unequivocal increases were observed in c-jun mRNA (U937 and HL-60) and protein (U937). In contrast, up-regulation of c-jun mRNA and protein in TAM67-expressing cells exposed to both PKC inhibitors was markedly attenuated relative to effects observed in parental U937 cells. Importantly, despite impaired up-regulation of c-jun at both the message and protein levels, TAM67-expressing cells were equally susceptible to PKC inhibitor-induced apoptosis as parental and empty vector U937 cells. Collectively, these findings raise the possibility that c-jun up-regulation in human myeloid leukemia cells undergoing PKC inhibitor-associated apoptosis represents a response to, rather than a cause of, apoptotic events. They further suggest that this phenomenon involves pathways that do not require PKC activation.

Characterization of alternatively spliced human SP-10 mRNAs.

Alternatively spliced mRNAs encoding the human intraacrosomal protein SP-10 were sought by the reverse transcriptase polymerase chain reaction (RTPCR). Eleven RTPCR products were identified, characterized, and found to represent authentic alternatively spliced SP-10 mRNAs. The 11 alternatively spliced SP-10 mRNAs encoded proteins ranging from 81 to 265 amino acids. The 10 smaller variants all resulted from one or two in-frame deletions in exons 2 and/or 3 of the SP-10 genomic sequence. Quantitative competitive RTPCR showed that the four largest SP-10 mRNAs represented the majority (> 99%) of the SP-10 message in testes from each of four men. The relative abundance of each of the four SP-10 mRNAs varied between individuals, but the longest SP-10 mRNA, SP10-1, which encoded a 265 amino acid protein, was consistently the most abundant, comprising 53-72% of the total SP-10 message. This was followed by the second largest SP-10 mRNA, SP10-2, which encoded a protein of 246 amino acids and comprised 15-32%. The third and fourth largest SP-10 mRNAs, SP10-3 and SP10-4, encoded proteins of 210 and 195 amino acids and accounted for 3.4-8.3% and 8.7-12.5% of the total SP-10 messages, respectively. The remaining 7 SP-10 mRNAs combined accounted for < 1% of the total SP-10 message. Within the low abundance group of mRNAs were two that deleted the entire third exon of SP-10. The present study suggests that phenomena of cryptic splicing and exon skipping occur within the SP-10 mRNA. Along with proteolysis, alternative splicing also helps to explain the heterogeneous forms of SP-10 that have been observed on Western blots of human sperm extracts.

Induction of apoptotic DNA fragmentation and c-jun downregulation in human myeloid leukemia cells by the permeant Ca2+ chelator BAPTA/AM.

The permeant Ca2+ chelator acetoxymethyl-1,2-bis(2-aminopheoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA/AM), an agent previously used to characterize drug-induced apoptosis in neoplastic cells, has been examined with respect to induction of DNA fragmentation and cytotoxicity in the human leukemia cell lines HL-60 and U937. Exposure of cells to various concentrations of BAPTA/AM for 6 h resulted in a biphasic induction of internucleosomal DNA cleavage, with maximal damage occurring at 10-microM concentrations. Higher BAPTA/AM concentrations were associated with the loss of internucleosomal cleavage products, but with the appearance of larger (i.e., 50-kilobase) fragments on pulsed-field gel electrophoresis. Cells exposed to 10 microM BAPTA/AM exhibited classic apoptotic morphology, whereas cells exposed to 50-microM concentrations displayed atypical features (e.g., cell swelling, chromatin clumping); in each case, substantial cytotoxicity was noted. The actions of BAPTA/AM did not depend upon the presence of extracellular Ca2+, nor were they affected by impermeant Ca2+ chelators. Measurement of cytosolic Ca2+ by Fura-2/AM or Indo-1 revealed late but not early increases in intracellular Ca2+ in BAPTA/AM-treated cells. Finally, BAPTA/AM-induced apoptosis was accompanied by the concentration-dependent downregulation of the immediate early response gene c-jun. These findings suggest a complex role for Ca2+ chelators such as BAPTA/AM in the regulation of human myeloid leukemic cell apoptosis, and indicate that this agent may selectively antagonize internucleosomal DNA fragmentation without interfering with other aspects of the apoptotic response and/or cell lethality.

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction.

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization.

Tissue specificity of the acrosomal protein SP-10: a contraceptive vaccine candidate molecule.

The tissue specificity of the intra-acrosomal protein SP-10 was examined by Northern blot and polymerase chain reaction (PCR) analysis. Messenger RNA from 36 tissues in the female baboon (Papio papio) was isolated, separated on agarose gels, transferred to nylon, and probed with either SP-10, beta-actin, or cyclophilin cDNA. Northern blots, which were processed at both low and high stringency, showed SP-10 to be expressed exclusively in the testis. The mRNA from each tissue was also reverse transcribed, and both SP-10 and beta-actin were amplified by PCR from the resulting cDNA. Ethidium bromide-stained agarose gels of the SP-10 PCR products showed three clear bands from the testis but no co-migrating bands from the other tissues. Southern blots of the PCR products showed that only the three bands in the testis were related to SP-10. The data demonstrate that the SP-10 gene products are testis specific, a characteristic essential for a contraceptive vaccine candidate molecule.

Cloning and sequencing of baboon and cynomolgus monkey intra-acrosomal protein SP-10: homology with human SP-10 and a mouse sperm antigen (MSA-63).

In this study, cDNAs encoding the intraacrosomal protein SP-10 were cloned and sequenced from baboon (Papio papio) and macaque (Macaca fascicularis) testis libraries and the sequence compared to that of human SP-10. Two alternatively spliced SP-10 cDNAs were obtained from both baboon and macaque testis libraries. The two cDNAs in each species contained open reading frames encoding proteins of exactly 285 and 251 amino acids. A 98% homology between baboon and macaque SP-10 was found at the protein and DNA levels. An 85% and 89% homology between baboon and macaque SP-10 and human SP-10 was present at the protein and DNA level, respectively. A mouse intraacrosomal protein, MSA-63, considered to be an SP-10 homologue, exhibited an overall 53% homology to nonhuman primate SP-10 and a 60% homology to human SP-10 at the protein level. Polymerase chain reaction analysis of testis mRNA confirmed the existence of two alternatively spliced SP-10 mRNAs in both nonhuman primates. Primer extension analysis indicated a common major transcriptional start site in baboon, macaque, and human SP-10 67 nucleotides 5' to the ATG codon. The amino acid sequence data for nonhuman primate SP-10s suggest that antibodies generated by vaccinating baboons and macaques with human SP-10 will likely recognize nonhuman primate SP-10, supporting the testing of an SP-10 contraceptive vaccine based on human SP-10 in these nonhuman primate models.