Human growth hormone gene transfer into tumor cells may improve cancer chemotherapy.

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor (NF)-kappa B via direct transfer of a gene encoding a negative dominant of its natural inhibitor I kappa B, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone (hGH) gene into human monocytic cell lines may also inactivate NF-kappa B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH-transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of I kappa B, hGH gene transfer diminished NF-kappa B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH-transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.

Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.

Peripheral blood T cells generated after allogeneic bone marrow transplantation: lower levels of bcl-2 protein and enhanced sensitivity to spontaneous and CD95-mediated apoptosis in vitro. Abrogation of the apoptotic phenotype coincides with the recovery of normal naive/primed T-cell profiles.

T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to >/=730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4(+) and CD8(+) cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (triangle upPsim) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA(+)/CD62-L(+)) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8(+)/CD45R0(+) subpopulation, although CD45R0(-) subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

Growth hormone prevents human monocytic cells from Fas-mediated apoptosis by up-regulating Bcl-2 expression.

Apoptosis and particularly Fas-mediated apoptosis has been proposed to play a key role in controlling monocyte homeostasis. We and others have documented the regulatory function of human growth hormone (hGH) on monocytic cells, which prompted us to investigate the role of hGH on their response to Fas antigen cross-linking. Using human promonocytic U937 cells constitutively producing hGH upon gene transfer and human primary monocytes cultured in the presence of recombinant hGH, we demonstrated that hGH diminished Fas-mediated cell death by enhancing the expression of the antiapoptotic oncoprotein Bcl-2 as well as the level of bcl-2alpha mRNA. In parallel, we established that overexpression of Bcl-2 through gene transfer into normal U937 cells also diminished Fas-induced apoptosis. Further, as a result of Bcl-2 overexpression, we found that hGH greatly depressed Fas-induced activation of the cysteine protease caspase-3 (CPP32), which in turn affected the cleavage of poly(ADP-ribose) polymerase. Altogether, these data provide evidence that hGH mediates its protective effect through a Bcl-2-dependent pathway, clearly a crucial step in enhanced survival of monocytic cells exposed to Fas-induced death.

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.

Relationship between proliferation and susceptibility to CD95- and CD2-mediated apoptosis in stimulated primary T lymphocytes: T cells manifesting proliferative unresponsiveness are preferentially susceptible to CD95-mediated apoptosis.

We examined the relationship between proliferation and susceptibility to Fas- and CD2-mediated apoptosis of human peripheral T lymphocytes that had been exposed in primary culture to CD3- or CD2-derived mitogenic stimuli in the presence of monocytes and exogenous IL-2. After 5 days, activated T cells were fractionated into large (F2) and small (F6) cells on Percoll density gradients and analyzed for their susceptibility to apoptosis and for their position in the cell cycle. Most F6 cells displayed a CD45RA+, CD25-, CD2R- phenotype and were unable to incorporate bromodeoxyuridine (BrdUrd) during the entire culture period. However, they were activated to express Fas Ag and some cell cycle regulatory proteins specific to late G1 phase. T cells with proliferative unresponsiveness were sensitive to Fas-mediated apoptosis whether it was triggered by anti-Fas mAb or by Fas ligand, but were almost completely resistant to CD2 apoptotic signaling. In contrast, F2 cells exhibited classical activation markers (CD45RO, CD25, and CD2R), had crossed S phase at least once, and were sensitive to both Fas and CD2 apoptotic signals. In large cells harvested earlier (on day 3), the signals were operative in both BrdUrd+ and BrdUrd- cells. Thus, S phase entry is not required for Fas- and CD2-mediated apoptosis. The profound proliferative unresponsiveness of F6 cells to CD3 and CD2 stimuli (bypassed by ionomycin plus PMA) and the CD2R- conformation of their CD2 molecules suggest that they may be in vivo anergized cells whose elimination, upon restimulation, is highly dependent on the Fas death pathway.

Apoptosis of immature thymocytes mediated by E2/CD99.

E2/CD99 is a 32-kDa transmembrane molecule that does not belong to any known family of proteins. It appears to regulate adhesion properties of T cells as previously reported, in particular, the induction of homotypic adhesion in CD4+ CD8+ thymocytes. Apoptosis induced via E2/CD99 displays characteristic morphologic features, but includes early mitochondrial alterations and phosphatidylserine exposure at the outer leaflet of the plasma membrane. It is not followed by detectable DNA fragmentation, and its time course is much longer than apoptosis induced via the Fas/CD95 pathway. It requires 18 h for completion. E2/CD99-induced apoptosis does not require any RNA or protein synthesis and still occurs following blockage of the Fas pathway. It is, however, dependent on CPP32 and IL-1beta-converting enzyme-type cysteine proteases, as shown by blockade with their respective specific inhibitors. This effect is restricted to double-positive thymocytes carrying an intermediate density of CD3 and including all CD69+ cells. Thus, E2/CD99 apears to mediate a distinctive apoptotic signal at a critical stage of thymocyte differentiation, i.e., when positive selection is known to occur.

Inhibitory effect of growth hormone on TNF-alpha secretion and nuclear factor-kappaB translocation in lipopolysaccharide-stimulated human monocytes.

Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use.

Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells.

Fas and CD2 receptors can transduce apoptotic signals through two independent biochemical pathways. In this study, we first evaluated the role of intracellular GSH in these signaling pathways by inducing variations in the GSH pool of activated peripheral T lymphocytes. Increasing the concentration of intracellular GSH by means of N-acetyl-L-cysteine (NAC) and GSH ethyl ester (OEt) resulted in total protection against cell death, while inhibiting GSH synthesis with buthionine sulfoximine (BSO) greatly enhanced cell sensitivity to Fas and CD2 apoptotic signaling. The protection exerted by NAC and GSH OEt was essentially based on their capacity to establish an intracellular reducing environment as it still occurred in BSO-treated cells. Thiol-containing compounds (cysteine, captopril, D-penicillamine and 2-mercaptoethanol) inhibited apoptosis while a series of non-thiol antioxidants (including catalase and vitamin E) failed to do so, suggesting that protection was secondary to thiols/disulfides exchange reactions at the level of cysteine residues in proteins and not to detoxification of reactive oxygen intermediates. This conclusion was further supported by the finding that no enhanced generation of O.-2 and H2O2 could be detected in cells experiencing early stages of apoptosis such as a decreased concentration of intracellular GSH and cell shrinkage. Also, protection occurred in the presence of protein synthesis inhibitors, indicating that it was due to post-translational sulfhydryl redox regulation of critical molecules involved in the apoptotic cascade. These data suggest that GSH, the most abundant intracellular thiol antioxidant, may be important in counteracting Fas- and CD2-mediated apoptosis of T lymphocytes.

Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA.

U937 cells lacking mitochondrial DNA (rho [symbol: see text] cells) are auxotrophic for uridine and pyruvate, hypersensitive to hypoglycemic conditions, and resistant to antimycin A-induced apoptosis. In spite of their obvious metabolic defects, rho [symbol: see text] cells possess a normal mitochondrial transmembrane potential, as well as near-normal capacity to generate superoxide anion after menadione treatment. Similarly to rho + controls, rho [symbol: see text] cells undergo apoptosis in response to tumor necrosis factor-alpha plus cycloheximide. Detailed comparison of the apoptotic process in rho + and rho [symbol: see text] cells reveals essentially the same sequence of events. In response to tumor necrosis factor/cycloheximide, cells first lose their mitochondrial transmembrane potential (delta psi m) and then manifest late apoptotic alterations, such as generation of reactive oxygen species and DNA fragmentation. Experiments involving isolated mitochondria from rho + and rho [symbol: see text] cells confirm that rho [symbol: see text] mitochondria can be induced to undergo permeability transition, a process thought to account for the pre-apoptotic delta psi m disruption in cells. Like rho + mitochondria, rho [symbol: see text] mitochondria contain a pre-formed soluble factor that is capable of inducing chromatin condensation in isolated nuclei in vitro. This factor is released from mitochondria upon induction of permeability transition by calcium or the specific ligand of the adenine nucleotide translocator atractyloside. In conclusion, it appears that all structures involved in the maintenance and pre-apoptotic disruption of the delta psi m, as well as a mitochondrial apoptotic factor(s), are present in rho [symbol: see text] cells and thus are controlled by the nuclear rather than by the mitochondrial genome. These findings underline the contribution of mitochondria to the apoptotic process.

CD2-induced apoptosis in activated human peripheral T cells: a Fas-independent pathway that requires early protein tyrosine phosphorylation.

Short-term activated peripheral T lymphocytes are susceptible to apoptotic cell death triggered by CD2 mAbs. The aim of this study was to examine whether the CD2-mediated pathway of apoptosis is linked to the Fas death pathway, as this is the case for CD3/TCR-triggered apoptosis in several models of T cells. Using T lymphocytes from patients harboring Fas gene mutations and displaying a profound defect in Fas signaling of cell death, we show that CD2- (but not CD3-) mediated apoptosis still proceeds normally. In normal activated T cells, CD3-mediated apoptosis is prevented by reagents that block the Fas/Fas-ligand interaction, namely soluble M3 (an antagonistic anti-Fas mAb) and soluble human Fas.Fc, a fusion protein able to bind released Fas-ligand. In contrast, CD2 signaling of apoptosis resists these blocking agents. Neither new protein synthesis nor the activation of calcineurin was required for CD2- and Fas-mediated apoptosis, suggesting that latent cytoplasmic "death" molecules were activated upon stimulation of the cells. In both cases, protein tyrosine kinases were transiently activated, as is exemplified by the autophosphorylation and exokinase activity of p56lck, yielding overlapping yet nonidentical profiles of protein tyrosine phosphorylation. Pretreating the cells with herbimycin A, before the addition of the apoptotic stimuli, almost completely inhibited CD2 transmembrane signaling of apoptosis, but left intact Fas-induced apoptosis. Our data suggest that CD2 is a Fas-independent cell death pathway that might contribute directly to the elimination of T cells expanding during an immune reaction.

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

Long-term persistence of transferred PPD-reactive T cells after allogeneic bone marrow transplantation.

T cells play an important role in protective immunity against tuberculosis. As patients are not reimmunized with BCG after BMT, the question arises as to whether PPD-specific memory T cells are transferred from the marrow donor to the recipient and persist in the long-term. We studied long-term survivors of non-T cell-depleted allogeneic bone marrow transplantation for in vitro PPD-induced proliferative responses (n = 14), and delayed-type hypersensitivity after intradermal injection of tuberculin (n = 20). We also studied 7 patients who received T cell-depleted bone marrow. Proliferative responses in the first group were low, but were increased by concentrating CD4+ T cells, the major responding cells in this system. In contrast, PBL from patients who received T cell-depleted marrow remained unresponsive to PPD, although they responded normally to CMV antigens. Of the 15 healthy patients in the first group who underwent tuberculin skin tests, 13 had positive reactions, while only two failed to react. (Five patients of the first group were suffering from chronic GVHD and 3 of them were negative.) All the patients in the second group had negative delayed-hypersensitivity responses. The difference between the two groups of patients was highly significant (P < 0.003). These results show that transferred PPD-specific T cells persist in long-term survivors of non-T cell-depleted BMT, even in the absence of reimmunization.

Substance P enhances IL-2 expression in activated human T cells.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

Induction of cytolytic function in resting peripheral blood CD8+/Leu-7+ T cells through IL2/p 75 IL2-receptor interaction: a study in the allogeneic human bone marrow transplantation model.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

Abnormally expanded CD8+/Leu-7+ lymphocytes persisting in long-term bone marrow-transplanted patients are resting pre-cytotoxic T-lymphocytes.

We analyzed the functional status of the small CD8+/Leu-7+ T-lymphocytes that circulate in increased proportions in the blood of many allogeneic bone marrow transplant (BMT) patients. Purified CD8+/Leu-7+ T cells were tested for their effect on T-cell proliferative responses. In contrast to CD8+/Leu-7-T-lymphocytes, such cells behaved as suppressor cells for lectin-induced mitogenic responses of the donor's peripheral blood lymphocytes. However, they did not interfere with the in vitro responsiveness to specific stimuli such as protein purified derivative (PPD) or alloantigens. We demonstrate that CD8+/Leu-7+ T cells are resting pre-cytotoxic T-lymphocytes (CTL) that can be induced by mitogenic lectins to express their cytolytic program in a non-specific, non-major histocompatibility complex-restricted manner against phytohemagglutinin-treated lymphoblasts or K562 target cells. The lectin-triggered cytotoxicity was achieved within a few days, together with limited cell division. Our results suggest that circulating CD8+/Leu-7+ T cells from BMT recipients are in vivo primed CTL awaiting cellular activation.