Effects of calcium depletion on human cells in vitro and the anomalous behavior of the human melanoma cell line MM170.

Seven strains of normal human cells (fibroblastic, skin epithelioid, and amniotic) ceased to proliferate in medium depleted of free calcium ion by titration with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), whereas the growth of 9 of 10 human melanoma cell lines was not affected. Fibroblasts showed a rapid drop in thymidine pool size and decreased incorporation of thymidine and uridine when treated with EGTA, followed during the next 48 hr by a decrease in plasma membrane potential and by development of a proliferative block in the G1 phase of the cell cycle. The calcium-independent melanoma line MM96 exhibited an early decrease in thymidine pool size and enhanced incorporation of nucleosides but continued to proliferate with little perturbation of the cell cycle or change in membrane potential. Tumor cell DNA may therefore be selectively labeled in the presence of normal cells. The anomalous, calcium-dependent melanoma line (MM170) showed an immediate increase in the thymidine pool size and in nucleoside incorporation and subsequently accumulated in G1 and G2 with diminution of membrane potential and of DNA and RNA synthesis. The proliferative block in MM170 cells could be reversed by addition of calcium ion or by replacement with control medium. Addition to the medium of all 8 nucleosides (50 microM), singly or together, did not prevent EGTA-induced cytostasis in fibroblasts or MM170; transport of thymidine across the cell membrane was enhanced by 24-hr EGTA treatment in fibroblasts, MM96, and MM170. Thus, although calcium affected thymidine utilization rapidly and differently in each of the three cell types, nucleoside starvation per se did not appear to be responsible for either type of proliferative block.

Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines.

Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.

Human gammadelta(+) T lymphocytes have in vitro graft vs leukemia activity in the absence of an allogeneic response.

Refractory acute lymphoblastic leukemia (ALL) is often incurable, and relapse rates following allogeneic bone marrow transplantation (BMT) remain high. We have reported that patients who develop increased numbers of gammadelta(+) T cells soon after BMT are significantly less likely to relapse. We now show in seven donor/recipient pairs that donor-derived Vdelta1(+)CD4(-)CD8(-)gammadelta(+) T cells are activated and proliferate in response to recipient primary ALL blasts. In addition, these cells have been shown to bind and lyse the recipient ALL blasts. Separately, gammadelta(+) T cells proliferate poorly or not at all in mixed lymphocyte culture against HLA-mismatched unrelated stimulator cells. These observations suggest that allogeneic gammadelta(+) T cells could be an effective immunotherapeutic strategy against refractory disease without the risk of graft-versus-host disease.

Silent polymorphisms within the coding region of human sodium/hydrogen exchanger isoform-1 cDNA in peripheral blood mononuclear cells of leukemia patients: A comparison with healthy controls.

We have examined the sequence of the cDNA encoding the sodium/hydrogen exchanger isoform 1 (NHE1), from 23 bases upstream of the start codon to 28 bases downstream of the stop codon. Template was prepared from (1) peripheral blood mononuclear cells (PBMC) isolated from 10 healthy unrelated Caucasian volunteers; (2) PBMCs isolated from 6 leukemic patients (acute lymphoblastic leukemia [ALL], n = 3; chronic lymphocytic leukemia [CLL], n = 1; chronic myelogenous leukemia [CML], n = 2); and (3) samples of 4 leukemic cell lines (ALL: CEM, MOLT4; AML: KG1a; CML: K562). NHE1 cDNA in normal PBMCs showed silent polymorphism of nucleotides 112 (N1: T, frequency 0.70; C, frequency 0.30; prevalence of heterozygosity 0.42); 2248 (N2: G, frequency 0.90; A, frequency 0. 10; heterozygosity 0.18); and 2493 (N3: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18). Deduced primary structure of NHE1 protein in all normal volunteers was identical to that previously published for NHE1 from renal and cardiac tissue. Similar to normal PBMCs, NHE1 cDNA from leukemic cells showed polymorphism of nucleotides N1, N2, and N3, but failed to demonstrate leukemia-specific sequence differences. We conclude that the coding region of NHE1 cDNA shows a greater level of polymorphism than is currently recognized, but that sequence mutation of NHE1 is not a key event in the pathogenesis of leukemia.

Solar and UVC-induced mutation in human cells and inhibition by deoxynucleosides.

Optimum conditions were established for quantitating the induction of hypoxanthine guanine phosphoribosyl transferase-deficient (HGPRT-) mutants in HeLa cells and in a human amelanotic melanoma cell line (MM96L). Compared at a fluence of equal toxicity (D37, fluence required to decrease cell survival to 37% of unirradiated control), noon sunlight in summer was slightly more mutagenic in MM96L than in HeLa cells (17 and 12 HGPRT- mutants per 10(6) survivors respectively). UVC (predominantly 254 nm) exhibited similar mutagenicity as equitoxic sunlight in HeLa but was 8-fold more effective in MM96L than equitoxic sunlight. Addition of a mixture of deoxyguanosine (20 microM), deoxyadenosine (20 microM), deoxycytidine (100 microM) and thymidine (20 microM) to the culture medium during the 7-day expression period following irradiation gave a 3-fold reduction in the UVC-induced mutation frequency of MM96L but not HeLa cells. The results suggest that these melanocytic cells are highly susceptible to mutagenesis by short wavelength UV, in a mechanism sensitive to deoxynucleosides.

Purine deoxynucleoside metabolism in human melanoma cells with a high spontaneous mutation rate.

A human melanoma cell line (MM96L) had a spontaneous mutation rate at the HGPRT locus of approx. 7 times normal. The cells had elevated dATP and dGTP pools, lacked purine nucleoside phosphorylase (PNP) and were sensitive to killing by deoxyadenosine, deoxyinosine and related purines but not to inosine or hypoxanthine. Four other melanoma cell lines exhibited a range of nucleoside sensitivities and dNTP pool sizes. Failure of intact MM96L cells to degrade exogenous deoxyadenosine and deoxyinosine to hypoxanthine was confirmed by NMR of culture medium. Normal melanocytes were PNP+ and were insensitive to deoxyinosine. Comparison of the metabolites of [14C]deoxyinosine from MM96L and a PNP+ cell line of similar doubling time (HeLa) showed that both cell types produced 14C-labelled guanine and adenine nucleotides, with [14C]dATP and [14C]dADP being found in MM96L. This indicates that human sAMP synthetase or a similar enzyme catalyses the conversion of dIMP to dAMP, the resultant elevation of dATP causing base misincorporation and a mutator phenotype.

Uptake and toxicity of safranine and triphenylmethylphosphonium ion in human tumour cells.

Despite having lower cell membrane potentials, four human melanoma cell lines and HeLa cells accumulated the membrane probe triphenylmethylphosphonium ion (TPMP, 2 microM) 2-10 fold more rapidly than human fibroblasts, and did not reach equilibrium during the 2 h incubation period studied. The difference was not due to drug toxicity, although at much higher levels of TPMP (greater than 20 microM) selective killing of melanoma cells compared with fibroblasts was observed. Some dependence of TPMP accumulation upon membrane potential was indicated by inhibition of uptake in fibroblasts depolarised by culture in calcium-deficient medium. Melanoma cells exhibited no change in TPMP uptake or membrane potential under these conditions. Uptake of safranine, an indicator of mitochondrial membrane potential, did not follow the TPMP pattern. Thus, the anomalous accumulation of TPMP by human tumour cells appeared to result from either carrier-mediated transport or rapid intracellular metabolism and could not be used to determine cell membrane potentials.

Cellular signaling events elicited by v-abl associated with growth factor independence in an interleukin-3-dependent cell line.

A temperature-sensitive mutant of the v-abl oncoprotein has previously been shown to have markedly reduced tyrosine protein kinase activity in interleukin 3 (IL-3)-dependent cells grown at restrictive (39 degrees C), compared to permissive (32 degrees C) temperatures. Transfection of this mutant v-abl into the IC2.9 cell line, generated the IC.DP subclone which was dependent on IL-3 for survival at 39 degrees C, but not at 32 degrees C. Furthermore, IC.DP cells cultured at 32 degrees C exhibited IL-3-independent thymidine incorporation, which was not apparent at 39 degrees C. Switching cells from the restrictive to the permissive temperature resulted in an increase in cellular inositol-1,4,5-trisphosphate, choline phosphate and diacylglycerol levels in the IC.DP cell line. These increases were only observed after a lag period of 4 h. Within 2 h of switching IC.DP cells previously maintained at 32 to 39 degrees C, there was a significant decrease in all three metabolites. Temperature switches had no effect upon these metabolites in the parent IC2.9 cell line. Down-regulation of protein kinase C inhibited v-abl-stimulated DNA synthesis in IC.DP cells cultured at 32 degrees C. IC.DP cells cultured at 32 degrees C were found to have a constitutively activated Na+/H+ antiport, although this activation was inhibited by the down-modulation of protein kinase C. These data indicate a role for phospholipid hydrolysis and protein kinase C activation in V-ABL-mediated abrogation of IL-3 dependence.

Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger.

The Na(+)/H(+) exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pH(i)). It is a ubiquitous, amiloride-sensitive, growth factor-activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pH(i) than normal hematopoietic tissue. We then show that a direct correlation exists between pH(i) and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na(+)/H(+) exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pH(i) and induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pH(i) decreases, apoptosis-measured by annexin-V and TUNEL methodologies-rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents. (Blood. 2000;95:1427-1434)

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines.

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.

Melphalan-resistant lymphoblastoid cell lines established from patients with ovarian cancer treated with cross-linking agents.

Lymphoblastoid cell lines (LCLs) were established from 83 patients with ovarian cancer by transformation of peripheral B lymphocytes with Epstein-Barr virus. Comparing the melphalan resistance of different groups of LCLs using the mean Do obtained from clonogenic survival assays, LCLs from melphalan-treated patients were significantly more resistant than LCLs from patients not treated with this drug. However, prior treatment of the patient with ionizing radiation was not associated with increased in vitro resistance of the LCL to this agent. In melphalan-treated patients where LCLs were established serially, the melphalan Do increased after further melphalan treatment in vivo and decreased when no further treatment was given. No correlation was found between age of donor and LCL resistance to any of the above agents. A group of 15 LCLs previously established from non-tumour donors was less resistant to melphalan than the LCLs from patients with ovarian cancer. In a group of 29 patients with advanced disease in whom the clinical response was known, LCL resistance to melphalan appeared to be associated with poor clinical response to cross-linking agents. These results suggest that B cell populations undergo long term, but not necessarily permanent, increases in resistance to melphalan.

Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers.

Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allogeneic stem cell transplantation is a cause of morbidity and mortality from viral pneumonitis. Antiviral drugs given to reactivating patients have reduced the mortality from CMV but have toxic side effects and do not always prevent late CMV disease. Cellular immunotherapy to prevent CMV disease is less toxic and could provide prolonged protection. However, a practical approach to generating sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is required. This study describes a system for generating sufficient CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 bone marrow transplant recipients from 200 mL donor blood. Donor monocytes are used to generate dendritic cells (DCs) in medium with autologous plasma, interleukin 4, granulocyte-macrophage colony-stimulating factor, and CD40 ligand. The DCs are pulsed with the immunodominant HLA-A*0201-restricted CMV peptide pp65(495-503), and incubated with donor T cells. These cultures are restimulated twice with peptide-pulsed lymphoblastoid cell lines (LCLs) or CD40-ligated B cells and purified with phycoerythrin (PE)-labeled pp65(495-503)/HLA-A*0201 tetramers by flow sorting, or with anti-PE paramagnetic beads. The pure tetramer-positive population is then rapidly expanded to obtain sufficient cells for clinical immunotherapy. The expanded CTLs are more than 80% pure, of memory phenotype, with a Tc1 cytokine profile. They efficiently kill CMV-infected fibroblasts and express the integrin VLA-4, suggesting that the CTLs could cross endothelial barriers. This technique is reproducible and could be used for generating CMV-specific CTLs to prevent CMV disease after allogeneic blood and marrow transplantation. (Blood. 2001;98:505-512)

In vitro generation of Epstein-Barr virus-specific cytotoxic T cells in patients receiving haplo-identical allogeneic stem cell transplantation.

Use of a partially mismatched related donor (PMRD) is an option for patients who require allogeneic transplantation but do not have a matched sibling or unrelated donor. Epstein-Barr virus (EBV)-induced lymphoma is a major cause of mortality after PMRD transplantation. In this study, we present a clinical grade culture system for donor-derived EBV-specific cytotoxic T cells (CTLs) that do not recognize haplo-identical recipient cells. The EBV-specific CTLs were tested for cytolytic specificity and other functional properties, including ability to transgress into tissues, propensity for apoptosis, degree of clonality, stability of dominant T-cell clones, and Tc and Th phenotypes. The EBV-specific CTLs were routinely expanded to greater than 80 x 10(6) over a period of 5 weeks, which is sufficient for clinical application. A CD8+ phenotype predominated, and the CTLs were highly specific for donor lymphoblastoid cell lines (LCLs) without killing of recipient targets or K562. Vbeta spectratyping showed an oligoclonal population that was stable on prolonged culture. The EBV-specific CTLs were activated (D-related human leukocyte antigen [HLA-DR+], L-selectin+/-) and of memory phenotype (CD45RO+). Expression of the integrin VLA-4 suggested that these CTLs could adhere to endothelium and migrate into tissues. The Bcl-2 message was upregulated, which may protect the CTLs from the apoptosis. The first demonstration of overexpression of bcl-2 in human memory CTLs. In addition, we show that lymphoblastoid cell lines used to generate CTLs are readily genetically modified with recombinant lentivirus, indicating that genetically engineered antigen presentation is feasible.

Influence of T cell depletion method on circulating gammadelta T cell reconstitution and potential role in the graft-versus-leukemia effect.

BACKGROUND: Our laboratory previously reported that leukemia patients who developed > or = 10% gammadelta+ T cells during the first six months after receiving an anti-TCRalphabeta T-cell-depleted (TCD) graft from a partially mismatched related donor (PMRD) had a disease-free survival (DFS) advantage. These gammadelta+ T cells were V81+CD3+CD4-CD8-CD69+HLADR+ and are cytotoxic to K562 cells. METHODS: In order to determine whether the anti-alphabeta TCD regimen was associated with these findings, we compared the reconstitution of gammadelta+ T cells from patients who received TCD PMRD grafts using the anti-TCRc4 MAb TIOB9-1A31 (previously reported) with similar patients who received grafts using the anti-CD3 MAb OKT3. RESULTS: Increased cytotoxic Vdelta1+ T cells were seen in 10 of 43 T10B9 TCD patients compared to 7 of 100 in the OKT3 TCD group (23% versus 7%, p = 0.010). T10B9 patients with increased gammadelta+ T cells also exhibited a higher range of increased gammadelta+ T cells and the length of time the gammadelta+ T cells remained high was longer when compared to OKT3 patients. Patients with increased gammadelta+ T cells whose grafts were T-cell depleted with T10B9 showed a significant decrease in relapse (p = 0.038). Similar rates and reduction in relapse were seen in OKT3 TCD patients, although significance was not reached due to the small number of patients with increased gammadelta+ T cells. Estimated 3 year disease-free survival was significantly improved in T10B9 patients with increased gammadelta+ T cells (0.79 versus 0.31, p = 0.009), a trend also seen in OKT3 patients (p = 0.091). DISCUSSION: These observations indicate that Vdelta1+CD4-CD8-cytotoxic T cells are associated with lower relapse rates and improved survival, and thus may have a role in a graft-versus-leukemia effect.