Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein.

At present, treatment of HIV infection uses small inhibitory molecules that target HIV protease; however, the emergence of resistant HIV strains is increasingly problematic. To circumvent this, we report here a new 'Trojan horse' strategy to kill HIV-infected cells by exploiting HIV protease. We engineered a transducing, modified, apoptosis-promoting caspase-3 protein, TAT-Casp3, that substitutes HIV proteolytic cleavage sites for endogenous ones and efficiently transduces about 100% of cells, but remains inactive in uninfected cells. In HIV-infected cells, TAT-Casp3 becomes processed into an active form by HIV protease, resulting in apoptosis of the infected cell. This strategy could also be applied to other pathogens encoding specific proteases, such as hepatitis C virus, cytomegalovirus and malaria.

Protein transduction: unrestricted delivery into all cells?

Several proteins can traverse biological membranes through protein transduction. Small sections of these proteins (10-16 residues long) are responsible for this. Linking these domains covalently to compounds, peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame fusions with full-length proteins, lets them enter any cell type in a receptor- and transporter-independent fashion. Moreover, several of these fusions, introduced into mice, were delivered to all tissues, even crossing the blood-brain barrier. These domains thus might let us address new questions and even help in the treatment of human disease.

In vivo protein transduction: delivery of a biologically active protein into the mouse.

Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.

Suppression of tumorigenicity in Wilms tumor by the p15.5-p14 region of chromosome 11.

Wilms tumor has been associated with genomic alterations at both the 11p13 and 11p15 regions. To differentiate between the involvement of these two loci, a chromosome 11 was constructed that had one or the other region deleted, and this chromosome was introduced into the tumorigenic Wilms tumor cell line G401. When assayed for tumor-forming activity in nude mice, the 11p13-deleted, but not the 11p15.5-p14.1-deleted chromosome, retained its ability to suppress tumor formation. These results provide in vivo functional evidence for the existence of a second genetic locus (WT2) involved in suppressing the tumorigenic phenotype of Wilms tumor.

The retinoblastoma protein and the regulation of cell cycling.

Increasing attention has been focused on how the retinoblastoma (RB) protein regulates cell growth. Recent evidence indicates that it is a substrate for phosphorylation by cyclin-dependent kinase-cyclin complexes and suggests that this phosphorylation modulates the ability of this protein to regulate transit through the cell cycle, perhaps in its G1 phase.

Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo.

The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G(1) cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G(1) cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G(0) quiescent cells and becomes hypophosphorylated (approximately 2 mol of PO(4) to 1 mol of pRB) in early G(1) and hyperphosphorylated (approximately 10 mol of PO(4) to 1 mol of pRB) in late G(1) phase. Here, we report that hypophosphorylated pRB, present in early G(1), represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G(0) and hyperphosphorylated pRB in late G(1) fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G(1) and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G(1). Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16(INK4a) gene, contained hypophosphorylated pRB that was bound to E2Fs in early G(1) and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G(1) cyclin-Cdk complexes.

Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo.

The protein transduction domain (PTD) embedded in the HIV TAT protein (amino acids 47-57) has been shown to successfully mediate the introduction of heterologous peptides and proteins in excess of Mr 100,000 into mammalian cells in vitro and in vivo. We report here that the modeled structure of the TAT PTD is a strong amphipathic helix. On the basis of this information, we synthesized a series of synthetic PTDs that strengthen the alpha-helical content and optimize the placement of arginine residues. Several PTD peptides possessed significantly enhanced protein transduction potential compared with TAT in vitro and in vivo. These optimized PTDs have the potential to deliver both existing and novel anticancer therapeutics.

Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1.

Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1.

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.

p73.

The discovery of p73 as a family member of p53 has instigated a number of studies in search of its function, regulation, and involvement in tumorigenesis. p73 has been identified as a transcription factor that can regulate p53-dependent transcriptional targets. Similarly to p53, p73 can induce apoptosis in response to various stimuli, including certain types of DNA damage. This evidence suggests that p73 may act as a tumor suppressor with overlapping functions of p53. While mutations of p73 appear rare in human tumors, some leukemias have shown silencing of the gene by hypermethylation. Thus, introduction of p73 into tumor cells possessing inactive p53 may provide a valuable therapeutic approach.

Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death.

Currently, delivery of expression vectors, proteins, and/or pharmacologically important peptidyl mimetics to target cells is problematic because of the low percentage of cells targeted, overexpression, size constraints, and bioavailability. Concentration-dependent transduction of full-length proteins and domains directly into cells would serve to alleviate these problems. Previous researchers have demonstrated the ability of proteins linked to the human immunodeficiency virus (HIV) Tat transduction domain to transduce into cells; but because of inefficiencies, this methodological potential has not significantly progressed since 1988. We describe, in this chapter, a significant increase in transduction efficiency of proteins and ease of use by (1) generation of a Tat protein transduction domain in-frame bacterial expression vector, pTAT-HA, and (2) development of a purification protocol yielding denatured proteins. We have transduced full-length Tat fusion proteins ranging in size from 15 to 115 kDa into approximately 100% of all target cells examined, including peripheral blood lymphocytes, all cells present in whole blood, bone marrow stem cells, diploid fibroblasts, fibrosarcoma cells, and keratinocytes. Transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 10 min. We conclude that our methodology generates highly efficient transducible proteins that are biologically active and have broad potential in the manipulation of biological experimental systems, such as apoptotic induction, cell cycle progression, and differentiation, and in the delivery of pharmacologically relevant proteins.

Composite tissue (limb) allografts in rats. III. Development of donor-host lymphoid chimeras in long-term survivors.

Eight LEW rat recipients possessing long-term-surviving (206-701 days) LBN vascularized hind limb allografts (CTAs) were tested for donor-host lymphoid chimerism. The recipients received various cyclosporine (CsA) treatment protocols in order to induce indefinite CTA acceptance. Histological examination of long-term-surviving CTAs demonstrated normal-appearing bone marrow in the donor limb. Lymphocytes isolated from host hemopoietic tissues (peripheral blood and/or spleen) by ficoll-hypaque density gradient centrifugation were tested against LEW-anti-BN antisera. Comparisons were made to standard curves employing various known concentrations of LBN and LEW cell combinations. The level of lymphocyte agglutination (dependent variable) showed a significant (P less than 0.025-0.005) linear relationship to the concentration of LBN donor cells (independent variable) present. Lymphocyte suspensions isolated from long-term CTA host peripheral blood and/or spleen showed a mean of 19.7% (+/- 9.7-95% confidence interval) donor LBN mononuclear cells present. Thus, it appeared that lymphoid cells originated from, and/or were released from LBN donor bone marrow into the circulation, resulting in chimeric repopulation of hemopoietic tissues. The presence of donor immunocytes in these limb allograft recipients may have been beneficial, and thus could have helped contribute to the long-term CTA survival observed.

Extensive prolongation of rat renal allograft survival following donor or nonspecific transfusions and concomitant immunosuppressant.

We report here a marked beneficial effect upon rat renal allograft survival transplanted across a strong histocompatibility barrier (BN----LEW) by pretransplant concomitant donor-strain blood transfusion (DST) and CsA treatment. Comparisons between recipient groups treated with pretransplant nonspecific blood (NST) and concomitant cyclosporine (CsA) or azathioprine (Aza) administration were also made. LEW recipients receiving only a BN renal allograft survived for a geometric average time of 8.9 days. Recipients receiving 1 ml of donor blood at weekly intervals, each week for three weeks prior to transplantation, demonstrated a geometric mean survival time (GMST) of 40.5 days. Recipients receiving this same regimen and concurrent CsA cover (5 mg/kg/day) starting 7 days prior to the first transfusion with discontinuation 5 days prior to transplantation showed extensive prolongation (greater than 100 days). Recipients treated with only CsA cover survived for a GMST of 34.4 days. LEW recipients receiving 1 ml of nonspecific blood at weekly intervals (DA, BUF, WKY, respectively) each week for 3 weeks prior to transplantation were prolonged to 27.7 days. Recipients treated with this same regimen while under CsA cover also demonstrated extended prolongation (greater than 100 days). Recipients receiving multiple donor blood transfusions under Aza (2 mg/kg/day) cover demonstrated lesser prolongation (22.8 days). Recipients receiving the multiple nonspecific blood protocol under Aza cover showed similar prolongation (38.6 days). Recipients treated only with Aza did not show prolonged survival (9.3 days). These differences in survival were considered significant among the 9 transplant groups as determined by ANOVA (P less than 0.001). The majority of recipient groups showed relatively poor renal function over their life spans, independent of whether prolongation occurred. Yet, renal function in the NST or particularly the DST groups covered by pretransplant CsA, demonstrated the best renal function in our laboratory over many years of investigations using the BN----LEW combination. In conclusion, there was a dramatic synergistic beneficial effect of prior multiple DST or NST specific to CsA, as opposed to another immunopharmacologic agent, Aza.

Biological methods for cell-cycle synchronization of mammalian cells.

Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Thus, many methods have been established to synchronize cells at specific phases of the cell cycle. Several of the common methods involve pharmacological agents, which act at various points throughout the cell cycle. Because of adverse cellular perturbations resulting from many of the synchronizing drugs used, other synchrony methods that involve less perturbation of biological systems, such as serum deprivation, contact inhibition, and centrifugal elutriation have a significant advantage. The advantages and disadvantages of these cell synchronization methods are discussed in this review.

Protein/peptide transduction domains: potential to deliver large DNA molecules into cells.

In vivo gene delivery can be achieved by direct injection of plasmid DNA. However, inefficient cellular uptake and nuclear import of plasmid DNA result in much lower levels of gene expression than observed when viral vectors are used as gene delivery agents. Recent studies have shown that transducing peptides, such as the HIV Tat protein, can carry large biomolecules from the extracellular environment directly into the cytoplasm and the nucleus of cells, both in vitro and in vivo. Thus, TAT-mediated transduction has the potential to increase the delivery of plasmid DNA to the nuclei of cells in vivo and thereby increase gene expression.