Linda Kelsey

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

In tumor cells that coexpress Cx43 and Cx26, the assembly of Cx43 is selectively impaired due to endocytosis. Assembly can be restored upon expressing a Cx43 sorting-motif mutant and mutants that cannot be phosphorylated on Ser-279 or Ser-282.

Retinoids regulate the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

Studies on optimal dose and administration schedule of a hematopoietic stimulatory β-(1,4)-linked mannan

Several complex carbohydrates have been found to significantly stimulate hematopoiesis. CARN 750, a polydispersed beta-(1,4)-linked acetylated mannan isolated from the Aloe vera plant, has been shown to have activity in wound repair, to function as a antineoplastic, and to activate macrophages. We report, herein, the hematoaugmenting properties of CARN 750 and its optimal dose and timing of administration in an animal model of irradiation-induced myelosuppression. We observed that subcutaneous injections of 1 mg/animal of CARN 750 had equal or greater stimulatory activity for white blood cell (WBC) counts and spleen cellularity as well as on the absolute numbers of neutrophils, lymphocytes, monocytes and platelets than did higher or lower doses of CARN 750 or an optimal dose of granulocyte-colony stimulating factor (G-CSF). Hematopoietic progenitors, measured as interleukin-3-supported colony forming units-culture (CFU-C) and high proliferative potential colony-forming cells (HPP-CFC) assays, were similarly increased by CARN 750 in the spleen but not in the bone marrow. The frequency of splenic HPP-CFCs and absolute number of splenic HPP-CFCs and CFU-Cs were optimally increased by 1 mg/animal of CARN 750. In contrast, bone marrow cellularity, frequency and absolute number of HPP-CFCs and CFU-Cs had as a dosage optimum 2 mg/animal of CARN 750. These parameters were similarly increased by G-CSF. In studies to determine the optimal protocol for the administration of CARN 750 we found that the hematopoietic activity of CARN 750 increased with the frequency of administration. The greatest activity in myelosuppressed mice was observed for all hematopoietic parameters except the platelet number in mice receiving daily administration of 1 mg/animal of CARN 750 with activity equal to or greater than G-CSF.

Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine

Liposomes and Flt3 ligand (Flt3L), a ligand for the fms-like tyrosine kinase receptor Flt3/ FLK2, can augment the immune response to an HIV peptide vaccine. The HGP-30 peptide used in these studies is a synthetic peptide that corresponds to a highly conserved region of HIV-1 p17 gag (amino acids 86-115). Mice were immunized with HGP-30 or HGP-30 conjugated to keyhole limpet hemocyanin (KLH) and delayed-type hypersensitivity (DTH) responses, antibody (IgG) amount and antigen-specific proliferative responses by spleen cells were used to monitor the immune response. Daily injections of Flt3L prior to HGP-30 administration enhanced significantly an antigen-specific lymphocyte proliferation response when compared with Flt3L, HGP-30 alone or HGP-30 containing liposomes. Intravenous administration of HGP-30 was superior to intramuscular (i.m.) immunization for the induction of DTH responses. The HGP-30/KLH containing liposomes enhanced both DTH and antibody responses, while liposomes containing HGP-30 peptide elicited only T cell responses. In these studies, either Flt3L or liposomes increased DTH responses compared with the i.m. injection of the HGP-30 vaccine alone.

Hematopoietic augmentation by a β-(1,4)-linked mannan

Abstract CARN 750 (injectable acemannan) is a polydispersed β-(1,4)-linked acetylated mannan isolated from the Aloe barbadensis plant. It has multiple therapeutic properties including activity in wound repair and as a biological agent for the treatment of neoplasia in animals as well as the ability to activate macrophages. We report herein that CARN 750 directly or indirectly has significant hematoaugmenting properties. We observed that the subcutaneous administration of CARN 750 significantly increases splenic and peripheral blood cellularity, as well as hematopoietic progenitors in the spleen and bone marrow as determined by the interleukin-3-responsive colony-forming unit culture assay and the high-proliferative-potential colony-forming-cell (HPP-CFC) assay (a measure of primitive hematopoietic precursors) in myelosuppressed (7 Gy) C57BL/6 mice. The greatest hematopoietic effect was observed following sublethal irradiation in mice receiving 1 mg CARN 750/animal, with less activity observed at higher or lower doses. Further, CARN 750, following daily injection, has activity equal to or greater than the injection of an optimal dose of granulocyte-colony-stimulating factor (G-CSF) in myelosuppressed mice. In this comparison, significantly greater activity was observed in the splenic and peripheral blood cellularity, and in the frequency and absolute number of splenic HPP-CFC as compared to the mice receiving G-CSF at 3 μg/animal. CARN 750, when administered to myelosuppressed animals. decreased the frequency of lymphocytes with a concomitant significant increase in the frequency of polymorphonuclear leukocytes (PMN). However, owing to the increased cellularity, a significant increase in the absolute number of PMN, lymphocytes, monocytes and platelets was observed, suggesting activity on multiple cell lineages. The latter is the primary difference in activity as compared to G-CSF which has activity predominantly on PMN.

In vivo haemoprotective activity of tetrapeptide AcSDKP combined with granulocyte‐colony stimulating factor following sublethal irradiation

We report that acetyl‐n‐Ser‐Asp‐Lys‐Pro (AcSDKP), which removes progenitor cells from cell cycle, in combination with granulocyte‐colony stimulating factor (G‐CSF) can significantly improve myelorestoration following irradiation (7 Gy). Peripheral blood, spleen and bone marrow (BM) cell recovery and progenitor cell reconstitution [IL‐3‐responsive colony‐forming cells (CFC) and high proliferative potential colony‐forming cells (HPP‐CFC)] were studied. Studies on the optimal schedule of AcSDKP administration revealed maximal effects on progenitor cells when AcSDKP was administered as a continuous infusion for 3 d starting 24 h prior to irradiation and used in combination with G‐CSF. The numbers of CFC and HPP‐CFC in the BM were significantly increased following irradiation in mice receiving AcSDKP and G‐CSF as compared to either drug alone. The numbers of CFC in the spleen were significantly increased in mice receiving AcSDKP and G‐CSF on days 10 and 14 as compared to AcSDKP alone, but not G‐CSF. Similarly, CFC and HPP‐CFC in the spleen were significantly increased in mice receiving AcSDKP and G‐CSF on day 18 as compared to mice receiving PBS and G‐CSF. These studies suggest that AcSDKP in combination with G‐CSF may have potential for the protection of progenitor cells in patients undergoing intensive chemo‐ and/or radiotherapy.

Adenovirus p53 purging for human breast cancer stem cell products.

Tumor cell (TC) contamination of stem cell products can contribute to relapse after high dose chemotherapy and stem cell rescue. A new purging technology using replication-deficient recombinant adenovirus (Adv) containing the p53 tumor suppressor gene (Adv-p53) has been suggested to reduce tumor contamination of autologous stem cell product. We demonstrate herein a safe and effective Adv-p53 purging procedure using four human breast cancer TC lines. Multiple parameters need to be achieved to successfully purge stem cell products, including a high cell:virus ratio, a small incubation volume, a long incubation time and 37°C rather than room temperature. These parameters are all interrelated and equally important for the inhibition of TC clonogenic growth. In our studies, we also observed that Adv could nonspecifically inhibit TC clonogenic growth, although Adv-p53 treatment led to a significantly greater inhibition of clonogenic growth by cells expressing mutated p53. The presence of peripheral stem cell (PSC) products was found to decrease the effect of Adv-p53 on TC clonogenic growth, suggesting that PSC products could compete with TC for infection by recombinant Adv. However, X-Gal staining after incubation with Adv containing-galactosidase demonstrated that PSC products were 2,000-fold more resistant to Adv infection than TC. We conclude that a 4-hour incubation of stem cell products (2 × 108/ml) with 4 × 1011 Adv-p53 particles is sufficient to completely purge TC with no effect on hematopoietic cell function.

Enhancement of Adenovirus-Mediated Gene Transfer to Human Bone Marrow Cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.

T-cell reconstitution by molecular, phenotypic, and functional analysis in the thymus, bone marrow, spleen, and blood following split-dose polychemotherapy and therapeutic activity for metastatic breast cancer in mice

We examined the effect of a maximum tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) on neutrophil and lymphocyte subpopulations in the peripheral blood leukocytes (PBLs), thymus, bone marrow, and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation (AuBMT) for breast cancer, suppressed both B- and T-cell populations and T-cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. We observed an organ- and phenotype-specific T- and B-cell recovery to normal levels following chemotherapy. However, despite normalization of cellularity and phenotype frequency, splenic lymphocytes remained unable to respond to normally concanavalin A (ConA). This polychemotherapy protocol in mice with an extensive experimental metastasis mammary tumor burden, was a dose lethal to 20% of the test group, which could be overcome with treatment by BMT and rHu interleukin (IL)-7. Furthermore, therapy with the T-cell augmenting agent rHu IL-7 had additive therapeutic activity and significantly prolonged survival beyond that of chemotherapy and BMT although it did not cure any mice with a heavy tumor burden. In summary, these studies demonstrate an organ-specific and selective polymorphonuclear neutrophil and T- and B-cell reconstitution following multidrug, split-dose chemotherapy on tissue and PBL populations, and a chronic depression in T-cell function, which when modified can result in significant therapeutic activity.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34 + cells

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor–mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte–macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC. Cancer Gene Therapy (2001) 8, 936–947