Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

Lymphokine-activated killer cell phenomenon. II. Precursor phenotype is serologically distinct from peripheral T lymphocytes, memory cytotoxic thymus-derived lymphocytes, and natural killer cells.

Culture of human peripheral blood lymphocytes (PBL) in partially purified and lectin-free interleukin 2 results in the generation of cytotoxic effector cells which have the unique property of lysing natural killer (NK)-resistant fresh human tumor cells. We have termed these effector cells "lymphokine- activated killer" cells (LAK). LAK are generated from both normal and cancer patients' PBL and are able to lyse both autologous and allogeneic tumor cells from all histologic tumor types tested. Our previous studies suggested that the LAK phenomenon was distinct from either the cytotoxic thymus-derived lymphocyte (CTL) or NK systems based on a variety of criteria. This study reports that the cell type involved is also distinct, as determined by phenotypic characteristics. The LAK effector cell phenotype was analyzed in parallel with alloimmune CTL, and LAK were found to be similarly susceptible to the monoclonal anti-T cell antibodies OKT-3 or OKT-8 plus complement. In contrast the LAK precursor was not susceptible to the OKT-3 or Leu-1 antibodies plus complement, while the ability to generate alloimmune CTL was totally obliterated when tested using the same PBL responder population; in fact, generation of LAK was found to be augmented five- to sixfold, clearly suggesting that LAK precursor cells are not T lymphocytes as defined by these antibodies. LAK precursors were found to be abundant in NK cell-enriched Percoll gradient fractions, which had been depleted of the 29 degrees C E- rosetting "high affinity" T cells. However, LAK precursors were found to be distinct from the majority of NK cells since lysis of fresh PBL with the monoclonal antibodies OKM-1, Leu-7, or OKT-11 significantly depleted or totally eliminated NK activity, while subsequent activation of the remaining cells generated high levels of LAK and in some cases augmented levels of LAK. LAK precursors were found to be distributed in the thymus, bone marrow, spleen, lymph node, and thoracic duct in addition to the PBL. Therefore, while the cell(s) responsible for activation and expression of LAK activity have some common features with the classic T cell-mediated CTL and NK cell systems, the LAK precursor cells are clearly distinct as determined by phenotype analysis using monoclonal antibodies and complement, and at present must be classified as a "null" cell.

Lymphokine-activated killer cell phenomenon. III. Evidence that IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells.

Purified interleukin 2 (IL-2) was found to be sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer (LAK) cells. The LAK activation factor was directly and consistently associated with IL-2 activity using classic protein purification techniques, adsorption to IL-2-dependent cell lines, and inhibition with anti-Tac antibody. As yet, no other cytokines have been found that perform the same role.

Biological activity of recombinant human interleukin-2 produced in Escherichia coli.

The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations. This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo. The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes. It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization. No functional differences between native and the recombinant interleukin-2 molecules have been detected.

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma.

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.

Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model.

BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) are common in human lung cancers. The wild-type form of p53 is dominant over the mutant; thus, restoration of wild-type p53 function in lung cancer cells may suppress their growth as tumors. PURPOSE: We investigated the therapeutic efficacy of direct administration of a retroviral wild-type p53 (wt-p53) expression vector (LNp53B) in an orthotopic human lung cancer model in nu/nu mice. METHODS: Proliferation of H226Br cells was determined by cell counting after infection with LNp53B in vitro. Irradiated (350 cGy) female BALB/c nu/nu mice were inoculated intratracheally with 2 x 10(6) H226Br cells (whose p53 gene has a homozygous mutation at codon 254) and treated beginning 3 days later with an intratracheal instillation of LNp53B retroviral supernatant for 3 days. RESULTS: Infection with LNp53B inhibited proliferation of H226Br cells in vitro. Thirty days after tumor cell inoculation, 62%-80% of the control mice showed macroscopic tumors of the right main stem bronchus. LNp53B suppressed H226Br tumor formation in 62%-100% of mice, and the effect was abrogated by dilution of the retroviral supernatant with inactive vector. CONCLUSIONS: Direct administration of a retroviral vector expressing wt-p53 may inhibit local growth in vivo of human lung cancer cells with abnormal p53 expression. IMPLICATIONS: Development of gene-replacement treatment strategies based on the type of mutations found in target cancers is warranted and may lead to the development of new adjunctive therapies and gene-specific prevention strategies for lung cancer.

Lymphokine-activated killer (LAK) cell phenomenon. IV. Lysis by LAK cell clones of fresh human tumor cells from autologous and multiple allogeneic tumors.

Activated killer cells are generated by the incubation of peripheral blood mononuclear leukocytes (PBL) in the lymphokine interleukin 2 (IL-2). Unseparated populations of these lymphokine-activated killer (LAK) cells lyse a variety of fresh noncultured human tumor targets, but they do not kill normal PBL. This study analyzed the generation and lytic specificity of LAK cell clones. Of 49 (84%) clones isolated by limiting-dilution techniques from a whole population of LAK cells, 41 manifested significant LAK cell activity. LAK cell clones had varied cell surface phenotypes. Clones with high and intermediate LAK cell activity were Leu 2+3-4+7-DR+Tac+ and Leu 2-3+4+7-DR+Tac+, respectively. Single LAK cell clones lysed multiple fresh human tumor targets including autologous sarcoma, 5 allogeneic sarcomas, and a colon cancer in addition to the cultured cell line K562. Autologous PBL were not lysed. Tumor targets were each lysed by multiple LAK cell clones. Sixteen subclones were derived from 5 of these LAK cell clones. These subclones had 99% or greater probability of being derived from a single cell. These subclones also exhibited lysis of multiple tumor targets. These findings suggest the existence of a shared determinant, expressed by multiple human tumors, which is recognized in common by multiple LAK cell clones.

Specific inhibition of interleukin 1 beta gene expression by an antisense oligonucleotide: obligatory role of interleukin 1 in the generation of lymphokine-activated killer cells.

The purpose of the studies reported here is to determine whether interleukin 1 (IL-1) plays an important role in the regulation of lymphokine-activated killer (LAK) cell induction. The addition of exogenous IL-1 to peripheral blood lymphocyte culture containing suboptimal concentrations of interleukin 2 (IL-2) resulted in induction of cytoplasmic pore-forming protein expression. Polymerase chain reaction results revealed that the mRNAs of both IL-1 alpha and IL-1 beta were induced within 6 h when cultured in IL-2 alone or in a combination of IL-2 and IL-1; however, tumor necrosis factor alpha and beta mRNAs were expressed earlier in peripheral blood lymphocytes stimulated with the combination of IL-1 and IL-2. Furthermore, we have examined the functional role of endogenous IL-1 in LAK activity. The lytic potential was significantly inhibited by an IL-1 receptor antagonist, which could block IL-1-mediated effects, or by specific neutralizing antibodies for IL-1, suggesting that the extracellular autocrine/paracrine pathway of IL-1 is involved in LAK activation. However, a synthetic IL-1 beta antisense oligonucleotide, which could specifically inhibit intracellular IL-1 beta protein expression as detected by Western blot, was more effective in reducing LAK killing, but it could not suppress the cytotoxicity generated by exogenous IL-1 plus IL-2. These findings clearly indicate the existence of an intracellular IL-1 autocrine circuit. Taken together, our results strongly indicate that IL-1 should be considered an obligatory factor in the regulation of IL-2-mediated lymphocyte functions.

Human interleukin 2 analogues that preferentially bind the intermediate-affinity interleukin 2 receptor lead to reduced secondary cytokine secretion: implications for the use of these interleukin 2 analogues in cancer immunotherapy.

Cancer patients undergoing interleukin (IL)-2-based immunotherapy frequently experience dose-limiting side effects believed to be caused by the actions of such cytokines as IL-1 beta, tumor necrosis factor (TNF)-alpha and -beta, and interferon-gamma (IFN-gamma). Human peripheral blood mononuclear cells (PBMC) or monocyte-depleted peripheral blood lymphocytes were stimulated for up to 7 days by either of 2 IL-2 analogues (R38A or F42K) that bind to the intermediate-affinity IL-2 beta gamma receptor but have reduced abilities to bind the high-affinity IL-2 receptor. We previously reported that these IL-2 analogues retain the ability to generate lymphokine-activated killing by PBMC. In this study, we analyzed the cytokine content of supernatants from stimulated PBMC and peripheral blood lymphocyte cultures by enzyme-linked immunosorbent assay. The secretions of IL-1 beta, TNF-alpha, and -beta, and IFN-gamma induced by either R38A or F42K were markedly reduced compared with secretions produced in response to recombinant wild-type IL-2. In 4 experiments, secretion was reduced an average of 39% for IL-1 beta, 57% for TNF-alpha, 83% for TNF-beta, and 86% for IFN-gamma. Polymerase chain reaction analysis of recombinant wild-type IL-2 or analogue-stimulated PBMC did not reveal the presence of IL-2 mRNA; thus, differential production of endogenous IL-2 could not account for these findings. These data suggest the interaction of IL-2 and the high-affinity IL-2 receptor on human PBMC or peripheral blood lymphocyte is required for maximal secretion of IL-1 beta, TNF-alpha, TNF-beta, and IFN-gamma. Because such cytokines are believed to mediate the toxicity seen with IL-2-based immunotherapies, IL-2 analogues with reduced binding to the high affinity IL-2 receptor may prove to be an effective and less toxic means of cancer treatment.

A rapid assay for stimulation of human lymphocytes by tumor-associated antigens.

Lymphocyte-stimulated protein synthesis (SPS) in response to human tumor-associated antigens was assessed by measuring [3H]leucine incorporation. Correlation of SPS with other in vivo and in vitro response was demonstrated by immunizing normal subjects with keyhole limpet hemocyanin and testing sequentially frozen lymphocytes and serum samples. One week after immunization, lymphocytes from normal subjects demonstrated increased SPS to keyhole limpet hemocyanin. This correlated with the appearance of delayed cutaneous hypersensitivity responses and preceded detection of hemagglutinating antibodies and increases in lymphocyte [3H]thymidine incorporation. There was no difference in the reactivity of fresh and viable frozen lymphocytes, and as few as 5X 10(5) lymphocytes/microtiter plate well could be used. Tumor-associated antigens were prepared from four lung carcinomas, six sarcomas, and six melanomas, using 3 M KCI extraction. Lymphocyte responses to both autologous and allogeneic tumor extracts were observed. Five of 15 patients demonstrated significant SPS to autologous tumor antigens. Fourteen of 20 lung cancer patients responded to lung cancer antigen, whereas only 11 of 41 patients with other tumors and 3 of 19 normal subjects reacted. Significantly, more lung cancer patients reacted to the tumor extract than to an extract of uninvolved lung from the same patient. Twenty-one of 42 melanoma patients responded to melanoma antigen. Ten of 33 patients with other tumors and 3 of 24 normal subjects reacted to the melanoma extract. Eight of 30 melanoma patients reacted to an extract of muscle from the same donor as was the melanoma antigen. Tumor-associated antigenic activity of 3 M KCI extracts can therefore be detected by measuring lymphocyte [3h]leucine incorporation.

Lysis of fresh human solid tumors by autologous lymphocytes activated in vitro with lectins.

Human peripheral blood lymphocytes (PBL), obtained from patients with a variety of cancers, were incubated in vitro with phytohemagglutinin, concanavalin A, and crude or lectin-free T-cell growth factors. The lectin-activated PBL of nine patients were capable of lysing fresh autologous tumor during a 4-hr 51Cr release assay. Multiple metastases from the same patient were equivalently lysed by these activated autologous PBL. No lysis of fresh PBL or lectin-induced lymphoblast cell targets was seen, although tumor, PBL, and lymphoblast cells were shown to be equally lysable using allosensitized cells. The activated cells could be expanded without loss of cytotoxicity in crude or lectin-free T-cell growth factors. The generation of cells lytic to fresh autologous tumor was dependent on the presence of adherent cells, although the lytic cell itself was not adherent. Proliferation was not involved in the induction of lytic cells since equal lysis was induced in irradiated and nonirradiated lymphocytes. Lectin was not required in the lytic assay, and the addition of alpha-methyl-D-mannoside to concanavalin A-activated lymphoid cells did not increase the lysis of fresh tumor cells. Activation by lectin for 3 days appears to be an efficient and convenient method for generating human cells lytic to fresh autologous tumor. These lytic cells may be of value for studies of the cell-mediated lysis of human tumor and possibly for tumor immunotherapy as well.

Tumor target binding induces phosphorylation of two M(r) 65,000 lymphokine-activated killer proteins.

Effector-target cell conjugate formation is an essential step during lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Protein phosphorylation changes in human LAKs after contact with NK-resistant (LAK-sensitive) tumors were examined by two-dimensional SDS-PAGE. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets led to increased phosphorylation of two M(r) 65,000 LAK proteins, pp65a and pp65b, with isoelectric points of 5.1 and 5.2, respectively. Phosphorylation of both substrates was initiated between 1 and 5 min after coincubation with tumor targets. Contact between LAKs and targets was required for p65 phosphorylation because soluble tumor factors failed to induce phosphorylation. Normal peripheral blood lymphocyte targets, which are bound very poorly by LAKs and are resistant to killing, failed to induce LAK p65 phosphorylation. The broad protein kinase inhibitor staurosporine inhibited phosphorylation of pp65a and pp65b, supporting the hypothesis that activation of a LAK protein kinase leads to p65 phosphorylation. Cross-linking of CD16 (Fc gamma RIIIA), which mediates antibody-dependent cellular cytotoxicity in LAKs, also led to increased pp65a and pp65b phosphorylation. Collectively, these data provide correlative evidence that p65 phosphorylation may be involved in the cytolytic function of LAKs.

Interleukin-2 or autologous lymphokine-activated killer cell treatment of malignant glioma: phase I trial.

In a Phase I study, recombinant interleukin-2 (IL-2) or autochthohous lymphokine-activated killer (LAK) cells were used to treat nine patients with malignant glioma. One patient received the combination of IL-2 and LAK cells. LAK cells were generated by culturing IL-2 with peripheral blood lymphocytes obtained from brain tumor patients. Escalating doses of LAK cells (10(8)-10(10) or recombinant IL-2 (10(4)-10(6) units) were administered by direct injection into the brain tissue surrounding the cavity left following operative tumor removal. There have been no signs of systemic or neurotoxicity following treatment. The tumor selective killing of the LAK cells used for these treatments was demonstrated by their ability to lyse glioma cells but not normal cells in vitro using a chromium release microcytotoxicity assay.

Variable expression of cytokines in human head and neck squamous cell carcinoma cell lines and consistent expression in surgical specimens.

Head and neck squamous cell carcinomas (HNSCCs) are associated with abnormal cell-mediated immunity at the primary tumor site. We investigated tumor-derived cytokines as factors underlying such abnormalities. Cytokine mRNA and protein of eight HNSCC-derived cell lines were tested; reverse transcription-PCR results indicated the presence of mRNAs for interleukin 1alpha (IL-1alpha) and transforming growth factor alpha (8 of 8); transforming growth factor beta and IL-1beta (7 of 8); and IL-4 and IL-6 (4 of 8). IL-2, IFN-gamma, and tumor necrosis factor alpha mRNA were not detected. Supernatants from six of these cell lines were analyzed by ELISA; IL-1alpha, IL-1beta, and IL-6 were found to be markedly increased compared to human papillomavirus-16-immortalized human oral keratinocytes. To determine whether the cell line findings are applicable to primary tumors, we performed immunohistochemical analysis on tumor specimens from 12 patients with invasive HNSCC. Universal intracellular production of IL-1alpha, IL-1beta, and IL-6 protein was detected. We conclude that the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients.

Synergy of tumor necrosis factor and interleukin 2 in the activation of human cytotoxic lymphocytes: effect of tumor necrosis factor alpha and interleukin 2 in the generation of human lymphokine-activated killer cell cytotoxicity.

Human lymphocytes can respond to interleukin 2 (IL-2) under serum-free conditions with generation of major histocompatibility locus-unrestricted oncolytic activity. This function has been named lymphokine activated killing (LAK). Although IL-2 is sufficient for the development of LAK, this function can be regulated positively by the addition of tumor necrosis factor alpha or beta (TNF-alpha or -beta). The cytotoxic synergy observed with TNF enables production of optimal LAK function at a 10-fold lower IL-2 concentration. Neither TNF-alpha nor -beta is able to induce LAK function in the absence of IL-2. Using TNF-alpha as a model, we demonstrate that (a) the cytotoxic synergy occurs with both fresh human tumors and cell lines; (b) the degree of IL-2/TNF-alpha synergy, for most peripheral blood lymphocyte donors, is dependent upon the IL-2 concentration used for activation with the most striking synergy observed at lower IL-2 doses; (c) synergy is specific for TNF-alpha and can be abrogated by neutralizing antibody against this cytokine; (d) addition of high-dose neutralizing antibody to IL-2 alone-stimulated peripheral blood lymphocytes can reduce the cytotoxicity capacity of these effectors suggesting an immunoregulatory role for endogenous TNF-alpha; and (e) TNF-alpha addition to IL-2-stimulated peripheral blood lymphocytes does not increase proliferation or cell recovery but does result in enhanced IL-2 receptor expression. Collectively, our results suggest that TNF-alpha (and -beta) have immunopotentiating roles in the amplification of non-major histocompatibility locus-restricted lymphocyte effector function.

Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells.

Peripheral blood mononuclear cells cultured in vitro with interleukin 2 (IL-2) become cytolytic towards both autologous and allogeneic tumor cells. We report here that IL-1 synergizes with IL-2 in serum-free conditions to produce increased (1.3-286-fold) lymphokine-activated killer (LAK) activity. The most dramatic synergy is seen with low IL-2 concentrations (10 U/ml, 222 pM) and 50-250 U/ml IL-1 alpha or beta. Kinetics of addition experiments demonstrate a specific requirement for IL-1 at or before addition of IL-2 to the culture. We postulate that one of the mechanisms whereby IL-1 augments LAK activity is by rendering LAK-precursors more responsive to IL-2. Up-regulation of the IL-2 receptor beta chain (Tac) and increased [3H]thymidine incorporation in cultures containing IL-1 and IL-2 support this view. In some instances, IL-1 alone is capable of maintaining/generating a small degree of cytolytic activity. Collectively, our data demonstrate that IL-1 is capable of interacting with low dose IL-2 to significantly augment LAK activity, potentially playing an important role in the early stages of LAK activation and differentiation. Because synergy is observed with dramatically reduced IL-2 concentrations, this system may offer an alternative approach to high dose IL-2 therapy for the treatment of neoplastic disease.

A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis.

Multicellular tumor spheroids approximate the three-dimensional configuration of primary and metastatic tumors. The effects of retrovirus-mediated transduction of wild-type p53 (wt-p53) were studied on multicellular tumor spheroids of human non-small cell lung cancer cell lines H322a, the p53 gene of which is homozygously mutated at codon 248, and WT226b, which has endogenous wt-p53. The growth of WT226b spheroids was not affected by exogenous wt-p53 transduction; the growth of H322a spheroids, however, was significantly inhibited by the addition of wt-p53 virus stocks. Transduction of cells by the wt-p53 retroviral vector and penetration of multiple cell layers in H322a spheroids was demonstrated by in situ polymerase chain reaction/hybridization with the neomycin-resistant neo probe. Apoptotic changes indicating programmed cell death were observed in H322a spheroids treated with the wt-p53 virus. These results suggest that retroviral vectors can penetrate into multiple cell layers of three-dimensional tumor masses and induce potentially therapeutic effects.

Characterization of the M(r) 65,000 lymphokine-activated killer proteins phosphorylated after tumor target binding: evidence that pp65a and pp65b are phosphorylated forms of L-plastin.

Contact between lymphokine-activated killer (LAK) cells and natural killer-resistant tumor targets SK-Mel-1 (human melanoma) or Raji (human lymphoma) stimulates phosphorylation of two M(r) 65,000 LAK proteins (pp65a and pp65b) with nearly identical isoelectric points. Phosphoamino acid analysis of pp65a and pp65b detected phosphorylation exclusively on serine residues. Phosphotyrosine could not be detected on either substrate after immunoblotting with an antiphosphotyrosine antibody, and herbimycin A treatment failed to inhibit p65 phosphorylation induced by target contact. However, phorbol myristate acetate treatment alone induced LAK pp65a and pp65b phosphorylation, suggesting phosphorylation may be mediated by protein kinase C or a protein kinase C-regulated kinase. The molecular weight and isoelectric points of pp65a and pp65b are similar to that reported for the human actin-bundling protein, L-plastin (L-fimbrin). On two-dimensional SDS-PAGE gel immunoblots, a peptide specific anti-L-plastin antiserum bound to pp65a and pp65b, suggesting that the phosphoproteins are similar or identical to L-plastin. In addition, two adjacent M(r) 65,000 LAK proteins were also detected by the antiserum and may correspond to unphosphorylated forms of L-plastin. On the basis of known properties of phosphorylated L-plastin, it is hypothesized that p65 phosphorylation in LAKs may regulate adhesion to tumor targets.

Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene.

Recombinant adenovirus-mediated transfer of the wild-type p53 gene into monolayer cultures or multicellular tumor spheroids of human non-small cell lung cancer cell line H358, which has a homozygous deletion of p53, markedly increased the cellular sensitivity of these cells to the chemotherapeutic drug cisplatin. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into H358 tumors s.c. implanted into nu/nu mice, followed by i.p. administration of cisplatin, induced massive apoptotic destruction of the tumors. These results support the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

Lysis of fresh and cultured autologous tumor by human lymphocytes cultured in T-cell growth factor.

Human lymphocytes derived from the peripheral blood of patients with a variety of cancers were grown in T-cell growth factor (TCGF) and tested in a 4-hr 51Cr microcytotoxicity assay against fresh and cultured autologous tumor, autologous cultured skin fibroblasts, and autologous fresh peripheral blood lymphocytes. Lymphocytes grown in TCGF caused significant lysis of autologous cultured tumor and fibroblasts but caused little lysis of fresh autologous peripheral blood lymphocytes in all of seven patients tested. This lytic activity against autologous cultured cells was not dependent on the source of serum used in culturing the lymphoid cells or the targets. Lymphoid cells grown in TCGF also were capable of causing selective lysis of fresh autologous tumor cells that had never been in culture in five of nine patients. Lymphoid cells growing in lectin-free TCGF caused selective lysis of autologous tumor in five of seven patients. These observations demonstrate that peripheral lymphoid cells grown in TCGF can be lytic for autologous cultured and autologous fresh tumor compared to the lysis of fresh autologous peripheral blood lymphocytes. The fact that these autoreactive cells, lytic for tumor, may be expanded to large numbers in TCGF suggests a possible role for these cells in studies of the control of the cytotoxic response of activated cells to tumor and possibly in the immunotherapy of tumors as well.