Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals.

BH3-only proteins function at a proximal point in a conserved cell death pathway by binding, through their BH3 domains, to other Bcl-2 family members and triggering mitochondrial events associated with apoptosis. Here, we describe a strongly pro-apoptotic BH3-only protein, designated Bbc3, whose expression increases in response to diverse apoptotic stimuli. bbc3 mRNA levels were induced by exposure to DNA-damaging agents and by wild-type p53, which mediates DNA damage-induced apoptosis. p53 transactivated bbc3 through consensus p53 binding sites within the bbc3 promoter region, indicating that bbc3 is a direct target of p53. Additionally, bbc3 mRNA was induced by p53-independent apoptotic stimuli, including dexamethasone treatment of thymocytes, and serum deprivation of tumor cells. Insulin-like growth factor-1 and epidermal growth factor, growth factors with broad anti-apoptotic activity, were each sufficient to suppress Bbc3 expression in serum-starved tumor cells. These results suggest that the transcriptional regulation of bbc3 contributes to the transduction of diverse cell death and survival signals.

Simulated lipoprotein transport in the wall of branched arteries.

Study of arterial blood flow dynamics improves our understanding of the development of cardiovascular diseases such as atherosclerosis. The transport and accumulation of macromolecules in the arterial wall can be influenced by local fluid mechanics. We used numeric simulations to investigate such transport in a T-junction model. Presumably an in vitro experiment would consist of gel segments inserted in the walls of a mechanical flow T-junction model near branch points where separation and recirculation zones are expected. The transport of low density lipoprotein (LDL) was investigated theoretically at these sites in a two dimensional numeric T-branch model. In the numeric model, the hydraulic conductivity of the porous gel wall segments was varied for a fixed species diffusivity to provide simulations with wall transmural Peclet numbers ranging from 0.3 to 30. Steady state flow patterns in the lumen of the two dimensional T-branch were simulated at Reynolds numbers of 250 and 500, using the software package FIDAP 7.61 to implement the finite element method. The simulations demonstrated that wall Peclet numbers greater than 1.0 were needed to achieve species concentration gradients within the wall that varied in the axial direction, thereby reflecting the influence of disturbed flow and pressure patterns in the lumen. As expected, the transmural concentration gradients were steeper when convection predominated. Blood flow in the lumen can influence the distribution of macromolecules in the arterial wall and needs to be investigated for the relevance to atherosclerosis.

Manometric changes during retrograde biliary infusion in mice.

The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [(14)C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 A in diameter to specific histological compartments.

Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155.

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-x(L) and related cell death suppressors. When BAD is phosphorylated on either Ser(112) or Ser(136), it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-x(L) at the mitochondria. Here we show that phosphorylation of a distinct site Ser(155), which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-x(L). Protein kinase A functioned as a BAD Ser(155) kinase both in vitro and in cells. BAD Ser(155) was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser(112)/Ser(136)- and a Ser(155)-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.

Role of the BH3 (Bcl-2 homology 3) domain in the regulation of apoptosis and Bcl-2-related proteins.

The Bcl-2 family of proteins play a prominent role in the regulation of apoptosis. From the initial identification of bcl-2 as an oncogene in follicular lymphoma through genetic studies in Caenorhabditis elegans to recent functional studies focusing on the importance of mitochondrial events in cell death signalling, the members of this protein family continue to be implicated in pivotal decision points regarding the survival of the cell. The family can be divided into two classes: those such as Bcl-2 and Bcl-xL that suppress cell death, and others, such as Bak and Bax, that appear to promote apoptosis. The Bcl-2 family is characterized by specific regions of homology termed Bcl-2 homology (BH1, BH2, BH3, BH4) domains, which are critical to the function of these proteins, including their impact on cell survival and their ability to interact with other family members and regulatory proteins. The identification of the BH3 domain as a potent mediator of cell death has led to the emergence of an additional family of proapoptotic proteins (such as Bad, Bik, Bid and Hrk) that share identity with Bcl-2 only within this death domain. These BH3-only proteins may be part of a regulatory network serving to integrate cell survival and death signals, an assertion that is supported by the identification of a BH3-only protein, Egl-1, as part of the central core of cell death signalling in C. elegans. While the mechanism of action of the BH3-only proteins remains unclear, recent studies on the regulation of critical protein-protein interactions and activity of Bad by phosphorylation in response to growth factor signalling suggest that the active state of BH3-only proteins may be regulated by post-translational modification. Additional modes of regulation, such as transcriptional, translational and subcellular localization, are also likely to be important.

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2.

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases.

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.

Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria.

Cytochrome c release and the mitochondrial permeability transition (PT), including loss of the transmembrane potential (Deltapsi), play an important role in apoptosis. Using isolated mitochondria, we found that recombinant Bax and Bak, proapoptotic members of the Bcl-2 family, induced mitochondrial Deltapsi loss, swelling, and cytochrome c release. All of these changes were dependent on Ca2+ and were prevented by cyclosporin A (CsA) and bongkrekic acid, both of which close the PT pores (megachannels), indicating that Bax- and Bak-induced mitochondrial changes were mediated through the opening of these pores. Bax-induced mitochondrial changes were inhibited by recombinant Bcl-xL and transgene-derived Bcl-2, antiapoptotic members of the Bcl-2 family, as well as by oligomycin, suggesting a possible regulatory effect of F0F1-ATPase on Bax-induced mitochondrial changes. Proapoptotic Bax- and Bak-BH3 (Bcl-2 homology) peptides, but not a mutant BH3 peptide nor a mutant Bak lacking BH3, induced the mitochondrial changes, indicating an essential role of the BH3 region. A coimmunoprecipitation study revealed that Bax and Bak interacted with the voltage-dependent anion channel, which is a component of PT pores. Taken together, these findings suggest that proapoptotic Bcl-2 family proteins, including Bax and Bak, induce the mitochondrial PT and cytochrome c release by interacting with the PT pores.

Quantification and pharmacokinetics of blood-brain barrier disruption in humans.

Hyperosmolar blood-brain barrier disruption (HBBBD), produced by infusion of mannitol into the cerebral arteries, has been used in the treatment of brain tumors to increase drug delivery to tumor and adjacent brain. However, the efficacy of HBBBD in brain tumor therapy has been controversial. The goal of this study was to measure changes in vascular permeability after HBBBD in patients with malignant brain tumors. The permeability (K1) of tumor and normal brain blood vessels was measured using rubidium-82 and positron emission tomography before and repeatedly at 8- to 15-minute intervals after HBBBD. Eighteen studies were performed in 13 patients, eight with glioblastoma multiforme and five with anaplastic astrocytoma. The HBBBD increased K1 in all patients. Baseline K1 values were 2.1 +/- 1.4 and 34.1 +/- 22.1 microl/minute/ml (+/- standard deviation) for brain and tumor, respectively. The peak absolute increases in K1 following HBBBD were 20.8 +/- 11.7 and 19.7 +/- 10.7 microl/minute/ml for brain and tumor, corresponding to percentage increases of approximately 1000% in brain and approximately 60% in tumor. The halftimes for return of K1 to near baseline for brain and tumor were 8.1 +/- 3.8 and 4.2 +/- 1.2 minutes, respectively. Simulations of the effects of HBBBD made using a very simple model with intraarterial methotrexate, which is exemplary of drugs with low permeability, indicate that 1) total exposure of the brain and tumor to methotrexate, as measured by the methotrexate concentration-time integral (or area under the curve), would increase with decreasing infusion duration and would be enhanced by 130% to 200% and by 7% to 16%, respectively, compared to intraarterial infusion of methotrexate alone; and 2) exposure time at concentrations above 1 microM, the minimal concentration required for the effects of methotrexate, would not be enhanced in tumor and would be enhanced by only 10% in brain. Hyperosmolar blood-brain barrier disruption transiently increases delivery of water-soluble compounds to normal brain and brain tumors. Most of the enhancement of exposure results from trapping the drug within the blood-brain barrier, an effect of the very transient alteration of the blood-brain barrier by HBBBD. Delivery is most effective when a drug is administered within 5 to 10 minutes after disruption. However, the increased exposure and exposure time that occur with methotrexate, the permeability of which is among the lowest of the agents currently used clinically, are limited and the disproportionate increase in brain exposure, compared to tumor exposure, may alter the therapeutic index of many drugs.

A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions.

Regulation of the cell death program involves physical interactions between different members of the Bcl-2 family that either promote or suppress apoptosis. The Bcl-2 homolog, Bak, promotes apoptosis and binds anti-apoptotic family members including Bcl-2 and Bcl-xL. We have identified a domain in Bak that is both necessary and sufficient for cytotoxic activity and binding to Bcl-xL. Sequences similar to this domain were identified in Bax and Bip1, two other proteins that promote apoptosis and interact with Bcl-xL, and were likewise critical for their capacity to kill cells and bind Bcl-xL. Thus, the domain is of central importance in mediating the function of multiple cell death-regulatory proteins that interact with Bcl-2 family members.

An in vitro flow model to study streaming during pelvic intra-arterial drug infusions.

Regional delivery of suitable drugs by intra-arterial infusion may offer a therapeutic advantage. High concentrations in the tumor are sought with reduced systemic toxicity. Adequate mixing of drug solutions with perfusing blood is essential to provide uniform distribution of drug to tumor-bearing tissue distal to the infusion site. Using a glass model of the iliofemoral and pelvic arteries, we have demonstrated that a streaming phenomenon occurs. Laminar "streamers" of slowly infused drug solution originate at the catheter tip and proceed nonuniformly into distal arterial branches. The intensity of streaming and the pattern of distribution are highly sensitive to catheter tip placement and quite unpredictable. The consequence of regional therapy under streaming conditions is severe maldistribution of drug in the infused tissues with potentially high levels delivered to normal tissues and simultaneous subtherapeutic levels delivered to tumor. Our in vitro model can be used to test appropriate infusion techniques that enhance mixing such as pulsed infusions and novel catheter designs.

Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins.

The survival-promoting activity of the Bcl-2 family of proteins appears to be modulated by interactions between various cellular proteins. We have identified a novel cellular protein, Bik, that interacts with the cellular survival-promoting proteins, Bcl-2 and Bcl-xL, as well as the viral survival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirus E1B-19 kDa. In transient transfection assays, Bik promotes cell death in a manner similar to the death-promoting members of the Bcl-2 family, Bax and Bak. This death-promoting activity of Bik can be suppressed by coexpression of Bcl-2, Bcl-XL, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik may be a common target for both cellular and viral anti-apoptotic proteins. While Bik does not show overt homology to the BH1 and BH2 conserved domains characteristic of the Bcl-2 family, it does share a 9 amino acid domain (BH3) with Bax and Bak which may be a critical determinant for the death-promoting activity of these proteins.

Induction of apoptosis by the Bcl-2 homologue Bak.

Cells are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide. Apoptosis comprises an intrinsic cellular defence against tumorigenesis, which, when suppressed, may contribute to the development of malignancies. The bcl-2 oncogene, which is activated in follicular lymphomas, functions as a potent suppressor of apoptosis under diverse conditions. Here we describe the complementary DNA cloning and functional analysis of a new Bcl-2 homologue, Bak, which promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. This raises the possibility that Bak is directly involved in activating the cell death machinery.

Different analogues of farnesyl pyrophosphate inhibit squalene synthase and protein:farnesyltransferase to different extents.

The inhibitory potency of farnesyl pyrophosphate analogues was investigated on two farnesyl pyrophosphate-consuming enzymes: squalene synthase, a secondary regulation site in the cholesterol synthesis pathway, and protein:farnesyl transferase, which plays a role in the function of Ras-proteins. For the transferase determination a rapid in vitro assay, using Sepharose-bound Ras-peptides, was developed. The distinct farnesyl pyrophosphate analogues showed a different order of potency in the inhibition of these two enzymes. Using the farnesyl transferase assay with pre-p21Ha-ras as substrate the same result was obtained. The difference observed in the in vitro assays was also reflected in the inhibition of cholesterol synthesis, protein prenylation in general and Ha-ras farnesylation in Rat-1.H-ras13 cells, a rat fibroblast cell line that overproduces human p21Ha-ras. This work shows that farnesyl pyrophosphate analogues can be developed for specific inhibition of different processes such as cholesterol synthesis and protein prenylation.

Paternal involvement after perinatal death.

This study describes paternal involvement after perinatal death in a large, mostly minority, lower socioeconomic status sample. Paternal presence at birth, holding the baby, and presence at a follow-up appointment were the indicators of paternal involvement. Perinatal death was defined as miscarriage, stillbirth, or neonatal death within 12 hours of birth. Data were collected from a retrospective chart review under the auspices of the Perinatal Mortality Counseling Program at Shands Hospital at the University of Florida, a tertiary care referral center in north central Florida. The sample included 722 cases of perinatal death between July 1978 and April 1991. The results indicated that many fathers experienced perinatal grief. More than half attended the birth, a quarter chose to hold their baby, and one fifth returned with the mother to follow-up appointments. Paternal-maternal cohabitation was the variable most predictive of the fathers' involvement after perinatal death. Race was predictive of the father being present at birth and at the follow-up visit, with white fathers more likely to be present than black fathers. Married fathers and those employed in a professional or manual labor occupation were more likely to be present at the birth than fathers who were unemployed, students, incarcerated, or in the military. Gestational age was predictive of the father holding the baby, with the likelihood increasing 1.04 times for each week of increase in gestational age. This study supports the need to include fathers in grief counseling, and the further investigation of the involvement of fathers when a perinatal death occurs.

Comparison of mechanical deformation properties of metallic stents with use of stress-strain analysis.

PURPOSE: Elastic and plastic deformation properties of the Wallstent, Palmaz stent, and Strecker stent were evaluated quantitatively with an in vitro model simulating forces exerted by an eccentric lesion. MATERIALS AND METHODS: A miniaturized compression testing device was constructed. Stress-strain graphs were obtained for each stent, and the elastic moduli and yield points were calculated. RESULTS: There is a 21-fold range in the elastic modulus among the Wallstent, Palmaz stent, and Strecker stents. The Palmaz stent was the only device to exhibit permanent plastic deformation. The 10-mm Palmaz stent will undergo 15% focal eccentric narrowing at 0.75 atm of pressure; the "standard braid" and "less shortening braid" 10-mm Wallstents at 0.55 and 0.25 atm, respectively; and the 10-mm tantalum Strecker stent at 0.08 atm. Overlapping of stents doubles the stiffness of the Wallstent and the Strecker stent and doubles the yield point of the Palmaz stent. The 4-9 mm Palmaz stent is 30% more resistant to deformation than the larger 8-12-mm version when expanded to identical 8-mm diameters. CONCLUSIONS: The "standard braid" version of the 10-mm Wallstent provides 2.3-fold additional strength for resistant stenoses compared with the "less shortening braid." Overlapping or nesting of stents may permit full expansion should there be incomplete expansion or recoil of a single stent. The 4-9-mm Palmaz stent is preferable from the standpoint of allowing the use of a smaller (7-F instead of 9-F) introducer sheath and also for providing superior resistance to deformation. A purely elastic stent such as the Wallstent is preferable in locations where permanent plastic deformation may occur, such as the thoracic outlet.

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

Injection characteristics and downstream contrast material distribution of flush aortography catheters: in vitro study.

Performance of 11 commercially available 4- and 5-F aortic flush catheters was evaluated with respect to the extent of upstream injection, catheter motion, and downstream homogeneity of a 10-, 15-, and 20-mL/sec bolus of 76% meglumine sodium diatrizoate at room temperature. Tests were made in a pulsatile aortic flow model containing circulating fluid isoviscous to blood. The injection process was recorded on videotape. Homogeneity of the contrast material bolus was determined spectrophotometrically from samples collected from the center and each of the four quadrants of the vessel lumen. Upstream contrast material injection between 1.5 and 7 cm in length emerged from all catheters; it was lowest with one of the "tennis racket" designs from one and a new spiral end-loop design (Halo) from another manufacturer. All catheters, except the most rigid and largest-caliber catheter (5.8 F) showed considerable shaft motion at the higher injection rates. Downstream contrast material mixing homogeneity was always best at the highest injection rate but altogether was better for the Halo catheter than for any other catheter tested. It is concluded that all tested 4- and 5-F aortic flush catheters show some undesirable features, but certain design modifications improve performance and comparative testing is helpful to distinguish such features.

Feedback inhibition of polyisoprenyl pyrophosphate synthesis from mevalonate in vitro. Implications for protein prenylation.

The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo.

Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.

The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p21ras, which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteine-aliphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatin-treated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.