Active immunotherapy of L1210 leukemia with neuraminidase-treated,drug-resistant L1210 sublines

Summary The effectiveness of neuraminidase-treated, drug-resistant L1210 sublines in active immunotherapy of L1210 leukemia was evaluated. Optimal conditions for the establishment of in vitro, drug-resistant cells included (a) proper drug concentration, (b) the use of logarithmic-phase cultures in fresh medium, containing 5 or 10% serum, and (c) continual exposure to drug. Active immunotherapy, after tumor burden was reduced with chemotherapy, with neuraminidase-treated cells alone was either effective or deleterious, depending upon the drug-resistant subline used for immunization. The combination of BCG and neuraminidase-treated cells was superior to treatment with chemotherapy only. Optimal response was observed with the use of parental L1210 cells, combined with BCG, in immunotherapy of parental L1210 tumor. The results emphasize that an important prerequisite to successful immunotherapy is that tumor vaccines must elicit immunologic products which are cytotoxic for residual tumor.Submitted in partial fulfillment of the requirements for the Masters of Science Degree, University of Oklahoma     

Structural Requirements for Cub Domain Containing Protein 1 (CDCP1) and Src Dependent Cell Transformation

Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.     

Fertile plant regeneration from cell suspension and protoplast cultures of barley (t Hordeum vulgare cv. Schooner)

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.     

Evaluation of time and dose in treating mammary adenocarcinoma with immunostimulants

Summary BCG, C. parvum, and reovirus were used as immunostimulants in treating murine mammary adenocarcinoma (A-10) after tumor burden had been minimized with BCNU. Immunostimulants were administered at different times with respect to chemotherapy. Different doses were used to determine the optimal response as measured by survival. BCG induced the best response when 6.67×10⁵ organisms were given 2 days after chemotherapy. The optimal response with C. parvum was observed after a dose of 0.35 mg was given 1 or 2 days after chemotherapy. Similarly, reovirus produced the best response when 10¹⁰ plaque-forming units were given 2 days after chemotherapy. These data are consistent with previous findings and support the notion that immunostimulants require an appropriate lymphoid substrate in order to induce an adequate anti-tumor response.Tge abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming unitsThis study was supported, in part, by Contract No. N01-CB-43864 and Grant No. CA 14460 from The National Cancer Institute     

Adherent indomethacin-sensitive suppressor cells in malignant melanoma

Summary Twenty-five patients with malignant melanoma were studied to determine the mechanisms underlying decreased cellular immunity in this disease. Sixteen patients were examined following surgical resections of tumor, and nine patients had evidence of metastases. Patients with metastatic disease (group II) had decreased lymphocyte transformation to concanavalin A (48,255±30,074) compared with controls (83,550±41,277) and patients free of disease (group I) 110,231±59,990) (P>0.01). Rigorous depletion of monocytes by adherent techniques resulted in an improvement in blastogenesis in seven of nine group II patients, whereas controls and group I patients had marked decreases in reactivity. Indomethacin also improved lymphocyte reactivity; a mean of 166.4% was recorded in five metastatic disease patients studied, as against a mean of 2% in group I and 9.65% in controls. In seven patients followed serially the presence of adherent suppressor cells tended to correlate with progressive disease, shorter survival, and decreased skin test reactivity.     

Dynamic Alignment Models for Neural Coding

Recently, there have been remarkable advances in modeling the relationships between the sensory environment, neuronal responses, and behavior. However, most models cannot encompass variable stimulus-response relationships such as varying response latencies and state or context dependence of the neural code. Here, we consider response modeling as a dynamic alignment problem and model stimulus and response jointly by a mixed pair hidden Markov model (MPH). In MPHs, multiple stimulus-response relationships (e.g., receptive fields) are represented by different states or groups of states in a Markov chain. Each stimulus-response relationship features temporal flexibility, allowing modeling of variable response latencies, including noisy ones. We derive algorithms for learning of MPH parameters and for inference of spike response probabilities. We show that some linear-nonlinear Poisson cascade (LNP) models are a special case of MPHs. We demonstrate the efficiency and usefulness of MPHs in simulations of both jittered and switching spike responses to white noise and natural stimuli. Furthermore, we apply MPHs to extracellular single and multi-unit data recorded in cortical brain areas of singing birds to showcase a novel method for estimating response lag distributions. MPHs allow simultaneous estimation of receptive fields, latency statistics, and hidden state dynamics and so can help to uncover complex stimulus response relationships that are subject to variable timing and involve diverse neural codes.     

THE EFFECT OF HYDROCORTISONE ON HeLa CELL GROWTH

HeLa Chessen cells have a doubling time of 18 hr when grown in MEM containing 10% calf serum and antibiotics. When hydrocortisone (1.7 μg/ml) is added to exponentially distributed cells in log growth in this medium, a new pattern of growth begins to emerge after 10–12 hr. This pattern is characterized by a transitional state lasting for about 6 hr, and then a new doubling time of about 35 hr is maintained thereafter. Hydrocortisone removes about 5% of the cells from the proliferative pool and extends the generation time of proliferating cells to about 30 hr. The extension of the generation cycle appears to occur almost entirely in late G₁. Cells grown as clones (average 6 cells/clone) prior to the addition of hydrocortisone, undergo these changes with doses as low as 0.00017 μg/ml of medium. When the average clone size is 1.5 cells per clone, the drug concentration must be 0.017 μg/ml or higher to initiate this response. The HeLa S₃ strain continues to grow with an 18-hr doubling time in the presence of hydrocortisone after a temporary delay in growth occurring between the 12th and 16th hour.