Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.

Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells.

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

Poly-L-lysine-based molecular conjugate vectors: a high efficiency gene transfer system for human progenitor and leukemia cells.

BACKGROUND: Targeted, specific receptor mediated gene transfer is a major goal of gene therapy research to accomplish gene transfer exclusively to the desired cell population. METHODS: First, the use of natural receptor for stem cell factor and transferrin receptor-targeted gene transfer using poly-L-lysine-based molecular conjugate vectors was evaluated in a panel of hematopoietic progenitor cell lines. Second, the ability of poly-L-lysine to enhance adenovirus mediated gene transfer efficiency was examined in different cell lines by using recombinant adenovirus-poly-L-lysine molecular conjugate conglomerates (recMCVEGFP). RESULTS: Despite effective ligand internalization receptor, gene expression amplification in receptor positive cell lines was not uniformly observed. Therefore, using a poly-L-lysine-based, receptor-targeted vector, neither transferrin nor natural receptor for stem cell factor mediated gene transfer can be considered a universally applicable procedure that exclusively depends on the presence of receptors on the cell surface; rather, it is a cell specific phenomenon. In our model, poly-L-lysine is the major contributor for gene transfer to hematopoietic progenitor cells, mediating the initial vector-cell binding. Human progenitor cell lines are poorly transduceable with recombinant adenovirus vectors. This new poly-L-lysine-modified, adenovirus-based vector could overcome virus tropism restrictions and consistently achieve very high transduction efficiency (>90%) in cells otherwise refractory to adenovirus gene transfer. CONCLUSIONS: Polylysine-based adenovirus vectors may have promise for situations in which high-efficiency gene transfer with transient high level transgene expression in hematopoietic cells is needed, such as leukemia vaccine protocols or for purging strategies in leukemia cell contaminated stem cell preparations.

Propylthiouracil reduces xenograft tumor growth in an athymic nude mouse prostate cancer model.

METHODS: Several anecdotal reports indicate that cancer may occasionally remain in a dormant state for prolonged periods in patients with hypothyroidism. Once the hypothyroid state is recognized and supplementation therapy with thyroid hormones is initiated, disease progression occurs. In this experiment, 6-n-propyl-2-thiouracil (PTU) was added to the water of athymic nude mice. The animals were subsequently inoculated with cells from a human prostate cancer cell line. RESULTS: The growth rate of subcutaneously implanted prostate xenografts was significantly slower in mice treated with PTU compared with mice that did not receive PTU. In a separate experiment, tritiated thymidine incorporation assays were performed in DU145 and PC3 human prostate cancer cells with and without PTU. No significant differences were observed, indicating that PTU did not exert any antitumor effect in vitro. CONCLUSIONS: Our study demonstrates that PTU inhibits the growth of human prostate tumors in nude mice via an indirect effect. This antitumor effect may be caused by hypothyroidism. This is the first in vivo study suggesting potential therapeutic applications for thyroid hormone manipulations in human cancer of the prostate. Further studies will determine growth kinetics of xenotransplanted prostate cancer in vitro and in vivo. PTU-induced hypothyroidism may be further explored in conjunction with other antineoplastic therapy.

Propylthiouracil-induced hypothyroidism reduces xenograft tumor growth in athymic nude mice.

BACKGROUND: Thyroid hormones are endocrine modulators of several vital processes that are crucial to tumor growth and differentiation. Several anecdotal reports in the literature suggest that some histologic types of carcinoma may remain in a dormant state for prolonged periods of time in patients with hypothyroidism, with eventual progression of the disease once the decreased thyroid function is identified and corrected. METHODS: Oral propylthiouracil (PTU) was used to induce hypothyroidism in athymic nude mice that were subsequently inoculated with lung adenocarcinoma and prostate adenocarcinoma cells. Mice were also treated with a combination of PTU and thyroxine, which resulted in hyperthyroid levels of T(4). RESULTS: Subcutaneous lung and prostate xenografts grew significantly more slowly in hypothyroid mice treated with PTU than in euthyroid or hyperthyroid mice, regardless of treatment with PTU. Tumors grew well in groups of mice that were changed from a hypothyroid state to a euthyroid state by withdrawal of oral PTU. Administration of PTU 3 weeks after tumor inoculation also caused the tumor growth to slow significantly compared with tumors in mice that did not receive PTU. Mice that received PTU and thyroxine had tumors that grew as well as the tumors in euthyroid control animals. CONCLUSIONS: Our study indicates that human lung and prostate tumors do not grow well in hypothyroid nude mice, and that rendering these animals euthyroid has a significant impact on the growth rate of these tumors. Furthermore, in vitro and in vivo data indicated that this was not a result of an interaction of the tumor cells with PTU, but rather a result of the hypothyroid state.

Treatment of thrombotic thrombocytopenic purpura with the cryosupernatant fraction of plasma: a case report and review of the literature.

We report a case of thrombotic thrombocytopenic purpura (TTP) that did not respond to extensive plasma exchange with fresh frozen plasma (FFP) but responded to plasma exchange with cryosupernatant. Several reports indicate that responses to cryosupernatant may be seen in patients refractory to FFP, and this approach may be appropriate in these patients before one recommends a splenectomy. The literature on refractory TTP treated with cryosupernatant exchange is reviewed.

Cloning and characterization of the prostate-specific membrane antigen promoter.

Prostate-specific membrane antigen (PSMA) is a protein that is expressed predominantly in normal prostate epithelial cells and in most adenocarcinomas of the prostate (Cap) and in virtually all Cap metastases. In this article we describe the cloning of a 2-kb human genomic DNA fragment containing the 5' upstream untranslated region of the PSMA gene and present evidence that it provides promoter activity. When the DNA fragment was cloned into transient expression vectors to examine promoter activity, the vectors were functional in promoting expression in several prostate and nonprostate cell lines in transient transfection assays. A 614-bp fragment derived from the 3' end of the 2-kb fragment may represent the minimal PSMA promoter as determined by deletion mutagenesis. The 2-kb fragment compared with the 614-bp fragment provided higher expression levels when using prostate-derived cell lines (DU 145 and LNCaP). The increased transcription using the 2-kb fragment was not as great in non-prostate cell lines. Little or no transcription over basal levels was seen with a 232-bp promoter fragment. When the concentration of dihydrotestosterone was depleted or supplemented in the growth medium, no significant effect was seen on PSMA-promoted transient expression in LNCaP cells, a prostate cell line. J. Cell. Biochem. 74:395-405, 1999. Published 1999 Wiley-Liss, Inc.

The effects of NSAIDs on breast cancer prognostic factors.

There are several risk factors involved in the pathogenesis of breast cancer. The role of non-steroidal anti-inflammatory drugs (NSAIDs) in the development of breast cancer has not been fully clarified. In order to investigate the impact of NSAIDs ingestion on prognostic factors of breast cancer we studied a total of 341 women with invasive carcinoma of the breast who presented between March and September 1993 to the Breast Cancer Clinic of the H. Lee Moffitt Cancer Center in Tampa, Florida. We noted that ingestion of NSAIDs was inversely associated with the size of the primary tumor, the lymph node status, and the number of involved axillary nodes. ingestion of NSAIDs may impact favorably on factors that determine the prognosis and clinical outcome of women with breast cancer.

The treatment of malignant mesothelioma with a gene modified cancer cell line: a phase I study.

Malignant mesothelioma is a tumor of the pleura for which there is no satisfactory treatment. It is almost universally fatal, regardless of the stage of the tumor at the time of diagnosis. Current treatment modalities include surgery, chemotherapy, and radiation therapy, although in some series none of these modalities is superior to no treatment at all. Because of the dismal prognosis for patients with malignant mesothelioma, a new mode of treatment is desperately needed. A promising area of research into the treatment of various malignancies is gene therapy. Recent studies have demonstrated the utility of exposing tumor cells to cells transduced to express the Herpes simplex virus gene for thymidine kinase (HSV-tk). By virtue of their expression of HSV-tk, the transduced cells are rendered susceptible to the antiviral drug, ganciclovir (GCV). and nearby tumor cells are killed by a phenomenon termed the bystander effect. In this protocol we propose a Phase I trial to study the safety and determine the maximal tolerated dose of an HSV-tk-transduced ovarian cancer cell line (PA1-STK cells) infused into the pleural cavities of patients with malignant pleural mesothelioma, followed by systemic administration of ganciclovir. The hope is that administration of ganciclovir will result in killing of the HSV-tk transduced ovarian cancer cells as well as the nearby malignant mesothelioma cells. This is a standard dose-escalation protocol.

Antitumor activity with the HSV-tk-gene-modified cell line PA-1-STK in malignant mesothelioma.

Malignant mesothelioma (MM) is a thoracic malignancy that is increasing in incidence. Since it is uniformly fatal and kills by local spread, investigators have proposed that MM is a good target for novel treatment approaches, such as gene therapy. We hypothesized that delivery of the HSV-tk gene, using gene-modified tumor cells (PA-1-STK cells), would result in an antitumor effect after treatment with ganciclovir. In in vitro mixing experiments, we found that PA-1-STK cells killed both mouse and human mesothelioma cells in a dose-dependent manner. Moreover, we found that PA-1-STK cells also prolonged survival of mice with MM when the percentage of total tumor cells was high (70%), but observed no survival benefit when the percentage of PA-1-STK cells was low (30%). These data support the rationale for a cell-based gene therapy approach to MM.

Gene therapy for malignant mesothelioma: a novel approach for an incurable cancer with increased incidence in Louisiana.

Malignant mesothelioma (MM) is a tumor of the pleura for which there is no satisfactory treatment. It is an almost universally fatal disease, regardless of the stage of the tumor at the time of diagnosis. Current treatment modalities include surgery, chemotherapy, and radiation therapy, although in some series none of these modalities is superior to no treatment at all. Because of the dismal prognosis for patients with MM, new modes of treatment are desperately needed. A promising area of research into the treatment of various malignancies is gene therapy. Recent studies have demonstrated the utility of exposing tumor cells to cells transduced to express the Herpes simplex virus gene for thymidine kinase (HSV-TK). By virtue of their expression of HSV-TK, the transduced cells are rendered susceptible to the antiviral drug, ganciclovir (GCV). Nearby untransduced tumor cells are killed by a so-called bystander effect. We are describing a Phase I clinical gene therapy trial for MM, which we are presently conducting at the Louisiana State University Medical Center of New Orleans. The purpose is to study the safety and to determine the maximal tolerated dose of an HSV-TK-transduced ovarian cancer cell line (PA1-STK cells) that is infused into the pleural cavities of patients. This infusion is followed by systemic administration of GCV. The hope is that administration of GCV will result in killing of both the transduced ovarian cancer cells as well as the nearby malignant cells.

Interaction of gemcitabine with paclitaxel and cisplatin in human tumor cell lines.

We have used clonogenic survival assays and flow cytometry of human lung A549, breast MCF7 and pancreas adenocarcinoma P-SW cell lines to examine the effects of gemcitabine (2'-deoxy-2', 2'-difluorocytidine) in combination with cisplatin, paclitaxel or radiation. Additive cell killing was observed for all cell lines when they were treated with cisplatin for 1 h followed by varying concentrations of gemcitabine for 24 h. Likewise, additive cell killing was noted in all cell lines when treated with gemcitabine for 24 h followed by varying doses of radiation. When A549 cells were exposed to gemcitabine for 24 h followed by a 1 h exposure to cisplatin, synergistic effects were noted. Using the latter regimen, MCF7 cells demonstrated additive cell kill, while the P-SW cells showed a more complex relationship with additive killing below 50 nM gemcitabine and less than additive effect above 50 nM gemcitabine. All three cell lines were also tested with various gemcitabine/paclitaxel combinations. When gemcitabine and paclitaxel were incubated concurrently, gemcitabine antagonized the cell kill produced by paclitaxel. All cell lines showed less than additive killing when either gemcitabine incubation preceded the paclitaxel incubation or the paclitaxel incubation preceded the gemcitabine incubation. Our results show that gemcitabine acts as a radiation sensitizer to increase the effects of radiation. Likewise, we demonstrate that the only uniformly beneficial drug combination schedule in all three cell lines was when cisplatin incubation preceded gemcitabine incubation. The gemcitabine/paclitaxel combinations were much more disturbing with respect to potential clinical trials. Our results would caution any planned clinical trials combining paclitaxel with gemcitabine to be reconsidered because of the potential for less than additive and even antagonistic effects of the combination.

Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA.

Early stage breast cancer.

Breast cancer is the most common malignancy seen in women. Due to the increasing awareness for this disease and the increasing use of screening mammography, more patients are now diagnosed with early stage disease. Over the past few years, the indications for adjuvant treatment have expanded considerably, and more women are now candidates for chemotherapy, hormonal therapy, or both. The role of various prognostic factors and their implications on the design of a multidisciplinary therapeutic approach is discussed in this review. Current recommendations regarding the extent of the surgery, the role of radiation, and its integration to the adjuvant treatment are discussed. Guidelines on adjuvant chemotherapy and adjuvant hormonal therapy are also being presented.

Phase II trial of 5-fluorouracil, leucovorin, interferon-alpha-2a, and cisplatin as neoadjuvant chemotherapy for locally advanced esophageal carcinoma.

BACKGROUND: Most patients with esophageal carcinoma present with locally advanced disease and a poor prognosis. Surgery or radiation provides palliation for locally advanced esophageal carcinoma. The role of neoadjuvant therapy remains to be defined. We administered neoadjuvant chemotherapy consisting of 5-fluorouracil (5-FU), leucovorin, interferon-alpha, and cisplatin to 11 patients with locally advanced disease. METHODS: Eleven patients with squamous cell or adenocarcinoma of the esophagus were treated peroperatively with two to three cycles of combination chemotherapy. Nine patients underwent resection with curative intent. RESULTS: Six patients received three cycles of chemotherapy, and five received two. Dose reduction was necessary for two patients. One patient achieved a pathologic complete response, histologically confirmed. Of the eleven patients, two did not undergo surgery because of progressive disease during chemotherapy. Seven of the 9 patients relapsed after surgery and 2 have been disease free for 27 months. CONCLUSIONS: The combination 5-FU leucovorin, interferon-alpha-2a, and cisplatin administered in a neoadjuvant setting resulted in a median survival of 11.8 months with a median time to relapse of 7 months.

Acute arterial thrombosis in a patient with breast cancer after chemotherapy with 5-fluorouracil, doxorubicin, leucovorin, cyclophosphamide, and interleukin-3.

BACKGROUND: Interleukin-3 (IL-3) is an experimental agent used to ameliorate neutropenia in patients receiving chemotherapy. Arterial thrombotic episodes after use of IL-3 have not been reported previously. METHODS: The case of a patient with Stage III adenocarcinoma of the breast who developed hypotension and acute cerebellar artery and superior mesenteric artery thrombosis after receiving chemotherapy and treatment with IL-3 is reported. RESULTS: To the authors' knowledge, this is the first patient with arterial thrombosis reported after treatment with IL-3. CONCLUSIONS: Interleukin-3 may be associated with increased propensity for thrombosis.

High expression of two diverged homeobox genes, HB24 and HB9, in acute leukemias: molecular markers of hematopoietic cell immaturity.

Homeobox genes encode for sequence-specific DNA-binding proteins which have been implicated in the control of gene expression during development and in certain adult tissues. Two recently characterized human homeobox-containing genes, HB9 and HB24, are known to be expressed in hematopoietic progenitors and to be involved in the regulation of growth and differentiation of progenitor cells to mature hematopoietic cell types. In this study, elevated levels of HB24 and HB9 mRNA expression were detected in bone marrow and peripheral blood mononuclear cells (PBMC) isolated from patients with acute myelogenous or acute lymphocytic leukemia. While the levels of both mRNAs were elevated in all the patients with acute leukemias, the levels of HB9 mRNA were more variable than those of HB24. Immunohistochemical analysis utilizing an HB24 polyclonal antiserum demonstrated elevated levels of HB24 protein in cytopreparations of acute leukemic cells. Nuclear run-on experiments showed that the increases of HB9 and HB24 mRNA transcripts in patients' cells were, at least in part, secondary to increased transcription. The expression of HB9 and HB24 correlated with the clinical status of the patient. No significant level of expression of either HB9 or HB24 was detected in PBMC isolated from patients in remission. In contrast to the findings with cells isolated from patients with acute leukemias, no significant increase in either HB9 or HB24 transcript levels were found in cells from patients with chronic lymphocytic or chronic myelogenous leukemia when compared to normal controls. These findings demonstrate that high levels of HB9 and HB24 expression are common features of acute leukemia and suggest the possibility that the dysregulated expression of these two genes may contribute to leukemogenesis. However, since these two genes are markers of immature hematopoietic cells they may not have an etiologic role in leukemogenesis.

Chromosome 21 rearrangement in acute biphenotypic leukemia.

A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documented to have a clonal chromosome 21 rearrangement, i(21q), four months before transformation to acute biphenotypic leukemia. For 4 months after transformation, isochromosome 21 persisted while the patient was receiving treatment with zidovudine. Vitamin D3 was added to zidovudine for an additional month, during which time the trisomy 21 clone reappeared as the predominant cell population. The unique aspects of this patient are the atypical evolution of chromosome 21, the transformation to biphenotypic leukemia, and the occurrence of i(21q) associated with biphenotypic leukemia evolving from an MDS.