Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.

Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (VH) gene probes. After obtaining the specific VH gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's VH DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS analysis. The myeloma cells stained positive with their own specific antisense VH biotinylated RNa probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific VH gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders.

Natural killer inhibitory substance produced by the peritoneal cells of patients with ovarian cancer.

Peritoneal cells obtained from 8 patients with minimal residual ovarian cancer produced a substance during in vitro culture that markedly inhibited the expression of natural killer (NK) cell-mediated lysis. Its molecular weight was less than 2,000, the same size as the NK-inhibiting substance (NK-IS), a similar NK-suppressive molecule produced by the peritoneal cells of rats. Human NK-IS suppressed the expression of antibody-dependent cell cytotoxicity as well as NK lysis, but it had no effect on erythrocyte-rosette formation and was not cytotoxic to peripheral blood lymphocytes or cell fractions enriched for large granular lymphocytes. NK-IS inhibited lysis mediated by interferon-activated lymphocytes and completely prevented NK activation when used in a preincubation. During intraperitoneal immunotherapy with Corynebacterium parvum, an agent that can activate peritoneal cytotoxic effectors, the production of NK-IS by peritoneal cells decreased considerably. Human peritoneal cells produce an NK-IS similar to the peritoneal cells of rats, and this material may create an environment within the peritoneal cavity that is permissive to the growth of NK-sensitive tumor cells.

Enhancement of natural killer cytotoxicity by cis-diamminedichloroplatinum (II) in vivo and in vitro.

To investigate the immunomodulating properties of cis-diamminedichloroplatinum (II) (CDDP), we studied the drug's effects on natural killer (NK) lymphocyte cytotoxicity. i.p. injections of CDDP (2-6 mg/kg) into adult mice significantly enhanced cytolysis of YAC-1 and K562 targets mediated by peritoneal and spleen cells. Lysis of the NK-resistant targets P815, EL-4, MOT, and RAJI was not increased, nor was the lysis of a YAC variant which had been specifically rendered resistant to NK lysis. Activated cytotoxicity was first noted 24 h after injection and returned to baseline by 7 days. Although i.p. injection enhanced peritoneal and spleen cell lysis, i.v. injection only activated spleen cells. Two analogues of CDDP, carbo- and iproplatin, effectively enhanced NK activity, but transplatin had no effect. Activated effector cells were non-adherent to nylon wool and serum-coated plates; they co-separated with lymphocytes on Percoll gradients, and they expressed asialo GM1 determinants. Incubation of targets with CDDP for 1 or 18 h significantly increased their sensitivity to lysis by normal murine spleen cells. These data indicate that CDDP has potent effects on NK cytotoxicity.

Role of inflammatory neutrophils in antitumor effects induced by intraperitoneal administration of Corynebacterium parvum in mice.

We studied the role of inflammatory neutrophils in the antitumor effects that follow i.p. injection of Corynebacterium parvum (1400 micrograms) into C3HeB/FeJ mice challenged with the murine ovarian teratocarcinoma. Peritoneal neutrophils, obtained from mice 6 hr after injection of C. parvum, exerted significant antitumor effects when injected admixed with murine ovarian terato-carcinoma cells into the peritoneal cavities of normal mice. Treatment of recipient mice with whole-body irradiation or repeated injections of silica prevented the antitumor effects, indicating that neutrophils were activating a second effector mechanism in recipient mice. Peritoneal cells obtained at 24 or 72 hr or at 7 or 11 days following C. parvum injection were considerably less effective in activation of this effector mechanism. Heat-killed C. parvum (6 hr)-induced neutrophils activated antitumor responses, but thioglycolate-induced cells were without effect. Antitumor responses in mice receiving peritoneal neutrophils were not due to simple transfer of C. parvum organisms in the inocula. These results indicate that inflammatory neutrophils, elicited into the peritoneal cavity by injection of C. parvum, play an important role in the activation of subsequent antitumor effects.

Synergistic effects of combination sequential immunotherapies in a murine ovarian cancer model.

The antitumor effects of Corynebacterium parvum in a murine ovarian teratocarcinoma model depend upon a sequential activation of neutrophils and macrophages within the peritoneal cavity. We studied the sequential administration of biological response modifiers that independently activate each phase of the response. Tumor-challenged mice treated by i.p. injection of a pyridine-extracted fraction of cell-free Propionibacterium acnes (PA-PE, 1400 micrograms) demonstrated prolonged survival in less than 20% of the cases. An i.p. injection of a detoxified Salmonella endotoxin (DSE) preparation (150 micrograms) had no effect on tumor outgrowth. However, i.p. treatment with PA-PE (1400 micrograms), followed by 150 micrograms of DSE 1 day later, resulted in long-term survival (greater than 100 days) in 40 to 60% of mice. This antitumor effect was only evident when PA-PE was administered first (before DSE) and optimal when DSE was administered 24 h after PA-PE. The synergistic antitumor effect could be duplicated when tumor-challenged mice were first treated i.p. with peritoneal polymorphonuclear leukocytes, elicited by injection of PA-PE, and then treated with DSE 18 h later. These data indicate that appropriately timed injection of biological response modifiers with complementary effects can result in a synergistic prevention of tumor growth.

Immunotherapy with biochemically dissociated fractions of Propionibacterium acnes in a murine ovarian cancer model.

The antitumor effect of two strains of Propionibacterium acnes (PAI and PAII) and chemically derived fractions from the whole bacterial cell were studied using a murine ovarian teratocarcinoma (MOT) model. When injected i.p. in high doses (700 to 1400 micrograms/mouse), both strains produce survival of a significant proportion of tumor-bearing mice (30 to 90%). On a weight to weight basis, however, PAI was significantly more effective than PAII. PAI and PAII were extracted using pyridine, which yielded four fractions, i.e., pyridine-extracted strains PAI and PAII (PA-PEI and PA-PEII, respectively) which are composed of the cell wall material extracted by pyridine, and the residues of PA-PEI and PA-PEII (PA-RI and PA-RII, respectively) which are composed of the residue material following the chemical extraction. The chemical composition of PA-PEI was different from that of PA-PEII (the latter had proportionately three times as many carbohydrates and one-third of the protein content of the former) and so were their antitumor properties in the MOT model. PA-PEI had markedly reduced antitumor effect when compared to the untreated cell on a per weight basis. Furthermore, curability was only seen when using a high dose (1400 micrograms/mouse). By contrast, the cell wall components extracted by pyridine from PAII (PA-PEII) had powerful antitumor effects, i.e., greater than 50% of mice given 1400-micrograms injections survived. The material contained in PA-PEII was further fractionated on the basis of its organic solubility in chloroform:methanol solvent. The water-soluble and solvent-insoluble fractions retained most of the antitumor effects of PA-PEII, while the water-insoluble and solvent-soluble fractions were only moderately effective, suggesting that the active moiety(ies) was associated with the nonlipid components of this fraction. Both residue fractions (PA-RI and PA-RII) were as effective on a per weight basis in controlling the growth of 10(5) tumor inoculum as were whole untreated cells. However, periodate oxidation of PA-RI resulted in complete loss of its antitumor effects. When surviving mice that had no evidence of tumor persistence following a tumor challenge (10(5) MOT cells) and i.p. treatment with PA were subsequently rechallenged with 10(4) tumor cells, survival was significantly prolonged, as compared to tumor-challenged (10(4) MOT) naive mice. In addition, 10 to 20% of these rechallenged mice had complete eradication of the tumor inoculum (no evidence of disease for greater than 120 days).(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple myeloma clones are derived from a cell late in B lymphoid development.

We have previously demonstrated that the immunoglobulin (Ig) heavy chain variable region (VH) sequences expressed by the malignant clone in multiple myeloma (MM) contain a high degree of somatic mutation without clonal diversity. This sequence can be used to identify all members of the malignant clone in this B cell malignancy. We sequenced the variable regions expressed by patients with MM and generated primers from the complementarity determining region (CDR) sequences specific for each patient's tumor. Using these primers, we performed PCR amplification on highly purified subpopulations of cells separated by expression of CD10, CD34 and CD38. The results of these experiments demonstrate: 1) there is a small fraction of CD10-expressing tumor cells in MM patients, 2) CD34-bearing malignant cells do not exist in MM, and 3) although the vast amount of tumor is in the CD38-expressing cells, a small amount of tumor is in the CD38-negative population. We also used these primers to determine whether pre-class switch (i.e., Cmu-expressing lymphocytes) clonal cells exist in these patients. After PCR amplification with CDR1 and Cmu primers, colony hybridization was performed using both framework 3 (FR3) and CDR3 probes. Out of > 200 FR3-hybridizing colonies, < or = 5 colonies also hybridized with the CDR3 probe. Colonies which hybridized with both these probes were sequenced, and none of these sequences matched even closely the CDR3 expressed by the malignant clone. These results make the existence of a pre-class switch malignant cell unlikely in MM. Overall, these results suggest that the malignant clone in MM derives from a cell late in B lymphocyte development.

A CD10-positive subset of malignant cells is identified in multiple myeloma using PCR with patient-specific immunoglobulin gene primers.

Immunophenotypic studies show the presence of CD10-bearing malignant cells in a small subset of multiple myeloma (MM) patients. We used a sensitive PCR-based technique in order to determine the frequency that MM patients contain a malignant subpopulation which expresses this antigen. The immunoglobulin (Ig) heavy chain variable region (VH) gene sequence expressed by the malignant clone in MM can be used as a tumor specific marker. After determining this sequence in six MM patients, patient specific VH oligonucleotide primers from complementarity determining region (CDR) sequences were generated. Bone marrow mononuclear cells from these patients were incubated with two different anti-CD10 antibodies or isotype identical murine IgG controls. Cells were then sorted by flow cytometry into the 1% brightest cells containing > 99.99% CD10-positive cells and two fractions including the 90 and 10% dimmest staining cells. PCR amplification was performed on DNA from approximately 10(4) cells (0.1 microgram) using patient specific CDR1 and CDR3 primers. Detectable PCR product was obtained in each sorted sample although the intensity of the band was much higher in cells lacking CD10 expression (the 90 and 10% dimmest fractions) than in the CD10-bearing (1% brightest) population. These results imply that there is a small population of CD10-bearing clonal cells in most, if not all patients with MM.

Deletion of genetic material from a poly(ADP-ribose) polymerase-like gene on chromosome 13 occurs frequently in patients with monoclonal gammopathies.

Recently, by using a probe for the nuclear DNA repair enzyme poly(ADP-ribose) polymerase gene, a pseudogene was found on the long arm of chromosome 13. RFLP analysis demonstrates the presence of a common "A" allele and a rare "B" allele, which has a deletion of approximately 200 bp. This deletion occurs more frequently in blacks than in whites in the United States. In two B-cell malignancies, Burkitt's and follicular lymphomas, there is a marked increased frequency of the expression of the B allele. Thus, we have analyzed the frequency of this allele in another B-cell malignancy, multiple myeloma (MM), which is also more frequently observed in blacks. We studied 97 patients with MM (41 black and 56 white patients) and 30 patients with the related disorder monoclonal gammopathy of undetermined significance (MGUS; 13 black and 17 white patients). The results demonstrate that the overall frequency of B allele expression (37%) is higher than in a noncancer control population (23%; P < 0.01). This difference is mainly due to the much higher frequency of B expression in black patients (52 versus 35% in black controls; P < 0.01), whereas there is no significant difference in white patients (18 versus 14% in white controls). Overall, B allelic frequency is similar in patients with MM and MGUS. Matched germline and tumor DNA show identical patterns of expression of these alleles. These results suggest germline B allelic expression predisposes one to MM and MGUS.

Primary mediastinal lymphoma in adults.

The incidence of primary mediastinal lymphoma in adults was investigated in 184 patients with non-Hodgkin's lymphoma. This entity was defined as disease within the mediastinum in patients who presented with symptoms due to an enlarging mediastinal mass. Of 184 patients, 17 presented with primary mediastinal lymphoma. All had a diffuse histologic pattern. The most common pathologic type was poorly differentiated lymphocytic lymphoma, diffuse (PDL-D), (11 cases). In nine of these 11 cases the patients had tumors of convoluted lymphocytes. The presentation was rapid in onset, with heart failure, pericarditis, dyspnea and superior vena caval syndrome predominating. Eleven of the 17 were clinical stage I or II, but eight of these had widespread disease on pathologic staging or rapid dissemination soon after diagnosis. In conclusion (1) primary mediastinal lymphoma is always diffuse in histology. (2) The most frequent pathologic type is PDL-D, with convoluted morphology. (3) Compression of vital intra-thoracic structures is common. (4) Although seemingly localized at presentation, this entity usually implies disseminated disease.

Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapheresis autografts.

Autologous transplantation is increasingly being used to treat patients with multiple myeloma (MM). Recently, peripheral blood progenitor cell (PBPC) harvest have been preferred over autologous bone marrow (BM) harvests due to reduced engraftment time, ease of attainment, and presumptive reduction of occult tumor involvement. To resolve this latter assumption quantitatively, we have used the unique immunoglobulin (Ig) heavy chain variable region sequence of the patient's myeloma cell as a marker of clonality. Samples from PBPC collections and 'back-up' BM harvests were obtained from 13 patients with MM and analyzed for tumor contamination using patient-specific oligonucleotide primers and the polymerase chain reaction. As expected, the percentage of tumor cells contaminating the BM harvest (median, 0.74%) was higher than in the PBPC specimens (median, 0.0024%). Because of the increased total number of cells required for PBPC transplantation, the increase in total number of contaminating cells in the BM vs PBPC autografts was less pronounced, (BM:PBPC tumor contamination ratios ranging from 0.9 to > 4500; median, 14). This confirms that in most but not all cases unmanipulated PBPC products are preferable over BM harvests as a method of reducing myeloma autograft tumor contamination.

Sulfur-containing amino acids decrease cisplatin cytotoxicity and uptake in renal tubule epithelial cell lines.

PURPOSE: Nephrotoxicity is one of the major dose-limiting side-effects of cisplatin (DDP). The disproportionate accumulation of cisplatin in kidney tissue may play an important role, however, therapeutic measures to prevent this prime cause of nephrotoxicity are not available. Because certain amino acids (AAs) have been reported to modulate DDP nephrotoxicity in vivo, we explored the potential of all 20 protein AAs, N-acetylcysteine and DL-homocysteine to reduce DDP cytotoxicity and uptake in S1, S3 (proximal tubule), and DCT (distal convoluted tubule) cell lines. METHODS: Immortalized but non-transformed renal tubule epithelial cell lines, derived from specific portions of the nephron of an SV40 transgenic mouse. were grown to confluency and exposed to various concentrations of DDP for 1 h with or without concurrent exposure to AAs in an otherwise AA-free Krebs-Ringer buffer (KRB). After 1 h, cell layers were washed and replenished with medium for cytotoxicity assays, or processed immediately for the determination of DDP accumulation. Cytotoxicity was assessed 48 h later by an MTT assay, and DDP uptake after 1 h was determined by atomic absorption spectroscopy. RESULTS: In an initial screening where the cells were concurrently incubated with 0.25 mM DDP and 1 mM AA for 1 h in KRB, only cysteine (Cys), methionine (Met), N-acetylcysteine and DL-homocysteine reduced DDP toxicity. This effect was enhanced at 5 mM AA and most potent for Cys, which reduced DDP cytotoxicity by 79 +/- 3% in S3 cells, by 78 +/- 12.2% in DCT cells, and by 19 +/- 3.6% in S1 cells (P < 0.05). Reduction of cytotoxicity was less for Met, DL-homocysteine, and N-acetylcysteine, in decreasing order. All four AAs also inhibited DDP uptake in renal cells, with Cys as the strongest inhibitor. Inhibition of DDP accumulation by 1 mM Cys after 1 h was 39% in S3 cells, 38% in DCT cells, and 28% in S1 cells. Again, reduction of uptake was less for the three other AAs. Pre-complexing of DDP with Cys for 16 h increased its uptake by 8- to 30-fold compared with native DDP, but markedly inhibited its toxicity. Thus, pre-complexing of DDP with Cys could not explain the reduced uptake of DDP, but could partly account for the reduction in cytotoxicity. Double-reciprocal Lineweaver-Burk plots of DDP concentration-versus-uptake rates at a constant concentration of Cys suggested that Cys competitively inhibited DDP uptake in S1 and DCT cells, and in a more complex fashion in S3 cells. CONCLUSIONS: We conclude that Cys, Met, N-acetylcysteine, and DL-homocysteine differentially inhibit DDP toxicity and uptake in cultured S1, S3, and DCT cells, and that the inhibition of uptake, as well as the complexation of DDP with Cys within the cell, may prevent toxicity. The structural element R-CH(NH2)-[CH2]1 2-S-R, which is common to all four molecules, may play a crucial role in blocking the transport of DDP, and could have future clinical applications.

Regulation of transforming growth factor-beta 1-mediated apoptosis in head and neck squamous cell carcinoma.

The apoptotic cell death in Cal-27 cells induced by exposure to transforming growth factor-beta 1 was inhibited by the endonuclease inhibitor aurintricarboxylic acid (ATA) in a concentration-dependent fashion. In vitro studies of cytotoxicity, DNA fragmentation, and protein synthesis by Cal-27 cell lines were performed. Inhibition of cytotoxicity as well as endonucleolytic DNA cleavage was detected. ATA did not inhibit cytotoxicity either via transforming growth factor cell-surface-receptor alteration or by inhibition of macromolecular synthesis. ATA-sensitive events occurred late during treatment. These data suggest that endonucleolytic DNA cleavage is a mandatory event leading to cell death in this system.

Suppressive factor or factors derived from head and neck squamous cell carcinoma induce apoptosis in activated lymphocytes.

Our laboratory has previously identified a soluble factor derived from head and neck squamous cell carcinoma that impairs lymphocyte proliferative responses in vitro. This study further investigates the nature of the interaction between these factors and T lymphocytes. The proliferative activity of phytohemagglutinin-stimulated peripheral blood lymphocytes and the Jurkat T-cell line was significantly suppressed (>50%) by the supernatants of 13 (41.9%) of 31 recently explanted head and neck squamous cell carcinoma samples. A characteristic morphologic appearance of these suppressed cells and ladderlike pattern of DNA fragmentation on gel electrophoresis indicated that the suppressive supernatants were inducing or predisposing T cells to apoptotic death. This apoptosis-inducing activity may be similar to that previously described in a suppressive supernatant obtained from an esophageal carcinoma cell line. These results shed further light on the mechanism behind a soluble immunosuppressive factor or factors produced by head and neck squamous cell carcinoma.

Successful immunotherapy with intraperitoneal Corynebacterium parvum in a murine ovarian cancer model is associated with the recruitment of tumor-lytic neutrophils into the peritoneal cavity.

The rejection of a murine ovarian teratocarcinoma (MOT) after i.p. injection of Corynebacterium parvum was investigated. Treatment with C. parvum (1400 micrograms) 24 hr after i.p. inoculation of a lethal number of tumor cells (10(5] induced an antitumor effect that cured 75 to 95% of the mice. Morphologic analysis and an in vivo cytotoxicity assay that measured the rate of disappearance of radioactivity from the peritoneal cavity after injection of 125IUdR-labeled tumor cells indicated that the antitumor effect was initiated during the first 24 hr after C. parvum injection. During this period of time, host effector cells retrieved from the peritoneal cavity prevented tumor growth in a Winn assay and lysed radiolabeled MOT targets in a 4-hr Cr-release assay. After separation of peritoneal inflammatory cells on a Percoll gradient, neutrophil-enriched fractions demonstrated significant in vitro tumor lysis, but neutrophil-depleted populations were ineffective. Microscopic analysis of lysis at the single cell level confirmed that neutrophils were binding to and lysing MOT targets. Further characterization of these tumor cytolytic neutrophils revealed that they are nylon wool-adherent, not generated in indomethacin-pretreated mice (but effectively generated in whole body-irradiated mice), and achieve lysis within 30 min after binding to MOT targets. These results indicate that neutrophils must be considered potential antitumor effectors that can be recruited by treatment with biologic response modifiers.

Direct cytotoxicity of polymorphonuclear leukocyte granule proteins to human lung-derived cells and endothelial cells.

Neutrophils, in the course of defending the host against microbial invasion, release a potent arsenal of proteins that can potentially damage host tissues. Defensins are major peptides of human polymorphonuclear leukocyte (PMN) granules and are both broadly microbicidal and cytotoxic to several tumor cell lines. To determine whether these peptides could play a role in neutrophil-mediated lung injury, we examined the cytotoxicity of defensins and other PMN granule proteins in a chromium release assay with human lung-derived cell lines MRC-5 (lung fetal fibroblast), A549 (lung adenocarcinoma with features of alveolar epithelium), and primary cultures of human umbilical vein endothelial cells (HUVEC). Crude fractionation of an acid extract of human PMN granules yielded four fractions A-D. Only fraction D (containing mostly defensins) was significantly cytotoxic to all three target cells. In contrast, fraction A (containing myeloperoxidase and lactoferrin) and fraction C (containing lysozyme) had little effect, and fraction B (containing chiefly cathepsin G and elastase) was only injurious to endothelial cells. The cytotoxicity of whole PMN granule extracts on pulmonary epithelial and fibroblast targets could be completely accounted for by their defensin content. Fraction D- and defensin-mediated cytotoxicity was concentration dependent, required at least 10 to 12 h to become manifest, and was inhibited by serum. The role of these peptides in lung damage during acute and chronic inflammation deserves further study.

Antigen-specific suppression of the in vitro cytotoxic response by a soluble factor produced by Corynebacterium parvum-induced allospecific suppressor cells.

The adjuvant Corynebacterium parvum, when administered intravenously during an ongoing alloimmunization, induces alloantigen-specific splenic suppressor cells which inhibit primary and secondary in vitro sensitizations. We have previously shown that these cells produce a soluble suppressor factor in culture. We now further characterize this factor and its mechanism of action. Release of this suppressive factor is dependent upon specific restimulation of the splenic suppressor cell with the sensitizing alloantigen for 24-48 hr in culture. The suppressor factor inhibits primary, but not secondary, in vitro sensitizations in an antigen-specific, genetically unrestricted manner. The suppressive activity is not absorbed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin. The factor does not lyse tumor cells bearing the sensitizing alloantigen. Delay in addition to primary cultures of as little as 4 hr after culture initiation leads to loss of suppressive activity, suggesting that this antigen-specific allosuppressor factor inhibits an early step in the sensitization of precursor cytotoxic T lymphocytes.

Defensins: antimicrobial and cytotoxic peptides of mammalian cells.

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including six invariant cysteines whose intramolecular disulfide bonds cyclize and stabilize them in a complexly folded, triple-stranded beta-sheet configuration. Generated by the proteolytic processing of 93-95 amino acid precursor peptides, the constitute > 5% of the total cellular protein in human and rabbit neutrophils (polymorphonucleated neutrophils--PMN) and are also produced by rabbit lung macrophages and by mouse and rabbit small intestinal Paneth cells. Despite their prominence in rat PMN, defensins are not found in murine PMN. The antimicrobial spectrum of defensins includes gram positive and gram negative bacteria, mycobacteria, T. pallidum, many fungi, and some enveloped viruses. Defensins exert nonspecific cytotoxic activity against a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. They appear to kill mammalian target cells and microorganisms by a common mechanism, which involves initial electrostatic interactions with negatively charged target cell surface molecules (likely the head groups of polar membrane lipids), followed by insertion into the cell membranes which they permeabilize, forming voltage-regulated channels. In addition to their antimicrobial and cytotoxic properties, some defensins act as opsonins, while others inhibit protein kinase C, bind specifically to the ACTH receptor and block steroidogenesis or act as selective chemoattractants for monocytes. Defensins are a newly delineated family of effector molecules whose contribution to host defense, inflammation, and cytotoxicity may be considerable for humans, even though it is unlikely to be revealed by experimentation with mice.

Mechanism of target cytolysis by peptide defensins. Target cell metabolic activities, possibly involving endocytosis, are crucial for expression of cytotoxicity.

In a previous study, potent tumor cytolysis mediated by human neutrophil peptide defensins occurred slowly over 3 to 15 h. Because these kinetics suggested a requirement for target cell metabolic processes before tumor killing could be realized, the mechanism of lysis by these purified peptides was further investigated. 125I-labeled defensin bound extensively to peptide-sensitive K562 targets with biphasic kinetics. Binding was inhibited in parallel with cytotoxicity when both assays were performed at low temperature or in the presence of FCS. The albumin content of serum could account for the inhibitory effects of FCS. Cytotoxicity was also antagonized by agents that interfered with target cell energy metabolism (azide and 2-deoxyglucose), the cytoskeletal apparatus (cytochalasin B and dihydrocytochalasin B), lysosomal function (NH4Cl and chloraquin), or calmodulin-mediated activities (trifluoperazine). FCS also completely removed membrane-bound defensin when it was added after 5 min of binding at 37 degrees C. However, significantly less defensin was removed when FCS was added at later time points after binding was initiated. Cytochalasin B and azide/2-deoxyglucose did not prevent binding of defensin to targets but it significantly inhibited the development of FCS resistance in membrane-bound peptide. However, these two classes of inhibitors acted during distinct time windows: cytochalasin-sensitive events were complete by 1 h, whereas azide/2-deoxyglucose continued to be inhibitory when added as late as 2 h after defensins. These latter data indicated that critical energy-dependent events continue after the cytochalasin-sensitive phase has been completed. The results suggest that defensin-mediated cytotoxicity requires initial binding of defensin molecules to targets and subsequent cytoskeletal- and energy-dependent translocation or internalization. Although the defensins are low m.w. peptides, the initial processes required for their cytotoxic activity resemble those of more complex bacterial, plant and mammalian cytotoxins.