Acid phosphatase cytochemistry of mitogen-transformed normal and chronic lymphocytic leukemia lymphocytes

Acid phosphatase cytochemistry was performed on lymphocytes stimulated in vitro with phytohemagglutinin, pokeweed mitogen, or concanavalin A. These electron microscopic studies demonstrated that activated lymphocytes from both normals and patients with chronic lymphocytic leukemia (CLL) had an increased number of lysosomes relative to resting cells. At the time of maximum thymidine incorporation, a reduced number of lysosomes was present in many transformed CLL lymphocytes, mainly medium-sized blast cells, in comparison to transformed normal cells. The findings demonstrate a lysosomal abnormality in phytomitogen transformed CLL lymphocytes which may be related to functional defects of these cells or to an incomplete transformation of a residual population of normal lymphocytes.     

Anti-T cell monoclonal antibodies in vivo. I. Inhibition of delayed hypersensitivity but not cutaneous basophil hypersensitivity reactions

As part of a study of the therapeutic potential of anti-T cell monoclonal antibodies, we studied the biologic effects of 8BE6, a mouse anti-guinea pig (GP) pan-T cell monoclonal antibody, on blood and tissue T cells and on the prototypic T cell-mediated reactions, classic delayed hypersensitivity (DH) and cutaneous basophil hypersensitivity (CBH). 8BE6 reacts to a 68,000 m.w. protein probably homologous with human CD5 (T1) and murine Lyt-1. A single dose of 1.8 to 3.4 mg 8BE6 caused lymphopenia and greater than 90% depletion of 8BE6+ peripheral T cells 1 to 72 hr later, and a significant but lesser decrease of lymphocytes reacting with another pan-T cell monoclonal antibody (p less than 0.02 at 24 hr). Free serum 8BE6 was detected for up to 48 hr after administration. Immunoperoxidase stains of tissue revealed that lymphocytes in lymph nodes and spleen were coated with mouse immunoglobulin 1 hr after antibody treatment and displayed in situ capping. Subsequently, there was a loss of T cells in all tissues (spleen, lymph node, liver, and kidney) except the thymus, with normal 8BE6 antigen staining returning by 72 hr. Areas of induration of DH reactions to PPD were reduced in 8BE6-treated GP, compared with pretreatment reactions in the same GP or in control-treated GP (p less than 0.001 for both). The numbers of infiltrating T cells and fibronectin-receptor-positive macrophages were also reduced. In contrast, 8BE6 had no effect on CBH reactions, as judged by erythema and basophil counts in 1-micron sections, although fewer T cells were found in reaction sites. There were no differences in IgM, fibronectin, or Ia staining between 8BE6-treated GP and controls. In vivo administration of a single dose of anti-T cell monoclonal antibody results in a transient, highly specific depletion of T cell populations in peripheral blood and tissues except the thymus. This treatment inhibits DH but not CBH reactions by systemic and local depletion of T cells.     

An Epstein-Barr virus transforming protein associates with vimentin in lymphocytes.

The Epstein-Barr virus (EBV) latent infection membrane protein (LMP) is likely to be an important mediator of EBV-induced cell proliferation, since it is one of the few proteins encoded by the virus in latent infection and since production of this protein in Rat-1 cells results in their conversion to a fully transformed phenotype. LMP was previously noted to localize to patches at the cell periphery. In this paper we examine the basis of LMP patching in EBV-infected, transformed lymphocytes. Our data indicate that LMP is associated with the cytoskeletal protein vimentin. Although LMP is fully soluble in isotonic Triton X-100 buffer, only 50% of it is extracted from cells in this solution. The rest remains bound to the cytoskeleton. LMP undergoes phosphorylation, and phosphorylated LMP is preferentially associated with the cytoskeleton. As judged by both immunofluorescence and immunoelectron microscopy, the vimentin network in EBV-transformed lymphocytes or EBV-infected Burkitt tumor lymphocytes is abnormal. Vimentin and LMP often colocalize in a single patch near the plasma membrane. In response to Colcemid treatment of EBV-infected cells, vimentin reorganizes into perinuclear rings, as it does in uninfected cells. LMP is associated with these perinuclear rings. Vimentin (or a vimentin-associated protein) may be a transducer of an LMP transmembrane effect in lymphoproliferation.     

Intrinsic defects explain altered proliferative responses of T lymphocytes and HVS-derived T-cell lines in gastric adenocarcinoma

We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.APV and MP-B should be considered as joint first authors     

转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes. Kinetics, cell-requirements and the role of recruitment

PPD-stimulated human peripheral blood lymphocytes have been shown to exert a nonspecific cytotoxic effect on allogeneic and xenogeneic target cells labeled with ⁵¹Cr. Chromium release induced by washed transformed lymphocytes was linear with time. At lymphocyte to target cell ratios of 120:1, significant killing could be demonstrated as early as $\text{1}\text{2}$ hr. At 8 hr, killing occurred with ratios as low as 3:1. The cytotoxic effect was related to the ability of the lymphocytes to transform to PPD, but reached an earlier peak than either DNA or RNA synthesis. Supernatants from lymphocyte cultures were not cytotoxic: instead, viable transformed cells were required. Partial removal of macrophages from the original cultures increased the cytotoxic effect.Lymphocytes could also be nonspecifically recruited to exert a cytotoxic effect by a mitogenic-like factor produced in transforming cultures. Preliminary evidence suggested that this factor acted independently of PPD and of histocompatibility antigens. These results are discussed with reference to possible amplification mechanisms for late cytotoxic effects, and to delayed hypersensitivity reactions in vivo.     

The improvement of amorpha-4,11-diene production by a yeast-conform variant

Aims:  To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast.
Methods and Results:  The ADS gene was mutated to the yeast-conform variant ADSm . The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells.
Conclusions:  Strains transformed with the yeast-conform allele ( ADSm ) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene.
Significance and Impact of the Study:  We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.     

Oncofoetal antigens of human trophoblast.

Rabbits immunized with human trophoblast cell membranes produced antibodies that were detected, by immunofluorescence, to react with normal human tissues, and, by complement-mediated cytotoxicity, with several transformed human cell lines. Absorption with trophoblast abolished all of these reactions, whereas multiple absorptions with lymphocytes, liver or kidney failed to remove reactivity with either trophoblast or certain transformed cells. To further identify the antigens responsible for these antibodies, rabbits were immunized with a chromatographed fraction of deoxycholate-solubilzed membranes prepared from KCl-extracted, ultracentrifuge-prepared trophoblast microvilli. The resultant IgG antibody reacted specifically with syncytiotrophoblastic membranes in sections of human placentae, in addition to recognizing the membranes of viable Chang liver, AV3, HEp-2, Sw/156 (kidney) and Sw/527 (breast) cells. That normal tissues, baboon or monkey placentae, and HeLa or Daudi cell lines did not react with this antibody, indicates the presence of species- and organ-specific antigens in human trophoblast, as well as the existence of trophoblast cross-reactive antigens on some transformed cells. The selective localization of these antigens at the interface of the materno-foetal graft suggests that they function biologically in the host-parasite relation of human pregnancy; their appearance on many transformed cells implies a similar function in the host-parasite relation of some human cancers.     

Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Distribution of surface charge and concanavalin a-binding sites on normal and malignant transformed cells

Measurement of the rate of agglutination with the positively charged poly- -lysine of normal lymphocytes, Moloney-virus-transformed lymphoma cells, normal fibroblasts and SV40 transformed fibroblasts, has shown that the normal cells were agglutinated at a higher rate than the transformed cells. The labeling density of cationized ferritin in electron micrographs of sectioned cells, also indicated a higher charge density for the normal lymphocytes and fibroblasts. The normal cells showed a more regular clustered distribution of cationized ferritin than the transformed cells, and pre-fixation of cells with glutaraldehyde before labeling with cationized ferritin resulted in a random distribution in both types of cells. The transformed cells had a higher agglutinability than the normal cells by Concanavalin A (ConA) and this difference was also found after treatment of the cells with neuraminidase. Labeling with ConA-ferritin showed the same distribution on the sectioned normal and transformed cells. The results indicate that there was a difference in the redistribution of surface charge by cationized ferritin in normal and transformed cells and that there was no detectable difference in redistribution of ConA-binding sites with ConA-ferritin.     

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.     

Haematological data for Matsumoto Eosinophilic Shinshu rats as determined by an automated haematology analyser

The Matsumoto Eosinophilic Shinshu (MES) rat originated from an inbred mutant colony of rats with spontaneous eosinophilia. As part of an investigation of the pathogenesis of the MES rat, we examined the haematology data for 106 males and 88 females and age-associated changes using an automated haematology analyser, flow cytometric analysis and morphological examination. The data at 10 weeks of age showed the MES rats had higher counts for eosinophils and neutrophils, slightly higher counts for lymphocytes, monocytes, basophils, and large unstained cells (LUCs), and slightly lower values for the erythrocytic parameters when compared with Sprague-Dawley (SD) rats. In data for MES rats aged 8 to 20 weeks, eosinophil counts increased with age up to 20 weeks together with some increased neutrophil counts. After 11 weeks of age, counts for lymphocytes, monocytes, basophils, and LUCs in the MES rats were also slightly increased. In female MES rats, flow cytometric analysis showed increased counts for pan-T+ cells, but blasts, abnormal granulocytes and lymphocytes were not detected morphologically. The MES rat characterized by the haematological findings could be a useful animal model for studies of hypereosinophilia.     

EMMA-4 analysis of iron in cells of the thymic cortex of a weaver-bird (Quelea quelea).

Combined morphological and analytical studies with the EMMA-4 analytical electron microscope have enabled very early erythroid cells to be identified within the cortex of enlarging thymic lobes of Quelea quelea. These early erythroid cells have pale cytoplasm (sometimes with ferritin-like crystals present), slightly pachychromatic nuclei and have fewer cell organelles (mitochondria) than lymphocytes. Counts for iron were approximately 70% lower than counts from mature erythrocytes found free in the cortex. Iron was also recorded from some epithelial reticular cells and pyknotic nuclei; no iron was recorded from small lymphocytes (thymocytes) in the cortex. The presence of very early erythroid cells is a further indication that erythropoiesis occurs in situ in the avian thymus.     

Herpesvirus saimiri U RNAs are expressed and assembled into ribonucleoprotein particles in the absence of other viral genes.

Marmoset T lymphocytes transformed by herpesvirus saimiri contain a set of five virally encoded U RNAs called HSUR1 through HSUR5. HSUR genes have been individually transfected into a nonlymphoid, nonsimian cell line (HeLa cells) in the absence of any other coding regions of the herpesvirus saimiri genome. The levels of HSUR1 through HSUR4 in HeLa transient-expression systems are comparable to those found in virally transformed T cells (23 to 91%). In contrast, HSUR5 is expressed at ninefold-higher levels in transfected HeLa cells. Immunoprecipitation experiments show that HSURs expressed in transfected cells bind proteins with Sm determinants and acquire a 5' trimethylguanosine cap structure, as they do in transformed T cells. HSUR1 or HSUR4 particles from transfected HeLa cells migrate between 10S and 15S in velocity gradients, identical to the sedimentation of "monoparticles" produced in virally transformed lymphocytes. We conclude from these transfection experiments that no other herpesvirus saimiri or host-cell-specific gene products appear to be required for efficient expression of the HSUR genes or for subsequent assembly of the viral U RNAs into small nuclear ribonucleoprotein particles. In lymphocytes transformed by herpesvirus saimiri, HSUR small nuclear ribonucleoprotein particles are involved in higher-order complexes that sediment between 20S and 25S. HSUR1, HSUR2, and HSUR5 dissociate from such complexes upon incubation at 30 degrees C, whereas the complex containing HSUR4 is stable to incubation.     

Short-term hyperglycemia induces lymphopenia and lymphocyte subset redistribution.

Alterations in lymphocytes are a common finding in both type I and type II diabetes. Autoimmune phenomena in type I diabetes, the stage of the diabetic disorder and metabolic effects of therapeutic interventions may also affect actual distribution of lymphocyte phenotypes. This study investigated immunological effects specific to standardized hyperglycemia in non-diabetic individuals to exclude immunological changes potentially related to diabetes stage and treatment. 37 subjects (mean age +/- SD 39 +/- 5 years) underwent a sequence-controlled crossover with oral administration of a solution containing either 75 g glucose or artificial sweetener (i.e. placebo). At rest and at two hours, counts of white blood cells (WBC), mixed lymphocytes, mature T-cells (CD3), T-helper cells (CD4), T-suppressor/ cytotoxic cells (CD8), B-cells (CD19), natural killer cells (CD16/CD56), and interleukin-2 receptor bearing peripheral blood mononuclear cells (CD25) were measured by flow cytometry. Subjects showed a significant decrease in WBC, lymphocytes, and all lymphocyte subsets with the OGTT compared with the placebo solution (p < .05 to p < .001). In non-diabetic individuals, short-term hyperglycemia induces immunological changes that may be relevant to explain similar findings in patients with diabetes mellitus. Future studies need to validate these findings and their potential clinical implications in a diabetic population.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.     

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultra-violet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m²-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01–1.0 μg/ml) for 3 different UV-A doses (3000–15000 J/m²). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks. These results suggest that the cytotoxic effects of photoactivated 8-MOP in human lymphoblastoid cells may depend on DNA interstrand crosslinks.     

Micro-Raman spectroscopy detects individual neoplastic and normal hematopoietic cells

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific, slow, biologically perturbing, or a combination thereof. Here, we show that single-cell micro-Raman spectroscopy averts these shortcomings and can be used to discriminate between unfixed normal human lymphocytes and transformed Jurkat and Raji lymphocyte cell lines based on their biomolecular Raman signatures. We demonstrate that single-cell Raman spectra provide a highly reproducible biomolecular fingerprint of each cell type. Characteristic peaks, mostly due to different DNA and protein concentrations, allow for discerning normal lymphocytes from transformed lymphocytes with high confidence (p < 0.05). Spectra are also compared and analyzed by principal component analysis to demonstrate that normal and transformed cells form distinct clusters that can be defined using just two principal components. The method is shown to have a sensitivity of 98.3% for cancer detection, with 97.2% of the cells being correctly classified as belonging to the normal or transformed type. These results demonstrate the potential application of confocal micro-Raman spectroscopy as a clinical tool for single cancer cell detection based on intrinsic biomolecular signatures, therefore eliminating the need for exogenous fluorescent labeling.