Cellular Biology and Urology

 

The Cellular Biology laboratory is divided into areas devoted to in-vitro cell culture, histopathology, molecular biology, electrophoresis, high pressure liquid chromatography (HPLC), microscopy and photography. The available equipment includes a state of the art laboratory computer (loaded with the most current software programs available for data analysis, word processing and statistical evaluations), a digital camera adapted to our microscope, and a new centrifuge for sample preparation. Techniques used in the laboratory include those basic for molecular biology studies, in situ hybridization, Western blots, and immunohistochemistry. We also perform tests to determine optimal drug administration protocols based upon agent toxicity, specific targeting ability and circulatory patterns.

The laboratory maintains stock cultures of cell lines for in vitro passage for the various projects. These cell lines were initially derived from cancers of the prostate (both androgen sensitive and insensitive lines), breast and brain (glioblastoma). Cells are serially passed under a laminar flow hood for immediate use, or frozen for future utilization at either -56oC in our REVCO freezer, or by immersion in liquid nitrogen (N2). For the in vivo evaluation of new pharmaceutical agents, in vitro cultivated tumor cells are implanted into mice or rats in the animal facility. Depending on the in vivo model employed, implantations of tumor may be either onto the flank or into an orthotopic location. Previous work and laboratory publications have promoted the use of orthotopic implantation as being preferential in the study and treatment of tumors. The natural (orthotopic) site is able to expose the implanted cells to a more normal amount of growth factors, nutrients and general environmental influences provided by surrounding tissues within this location. Orthotopic implantations have been performed in both the prostate and brain tumor models we use.

Development and testing of new molecular therapeutic agents

Two antisense oligonucleotides (oligos) are under evaluation for the treatment of several types of malignancies, and each has been granted a United States Patent. The first patent was issued on March 11, 1997 (#5,610,288) and includes the complementary nucleotide base sequence for mRNA encoding the human epidermal growth factor receptor (EGFR). The second, which was issued April 6, 1999 (#5,891,858), includes the complementary base sequence for mRNA encoding transforming growth factor-a (TGF-a) which binds to the EGFR. These are the only patents issued to the Hektoen Institute in its almost 60 year distinguished history. Together, these protein targets form an autocrine loop which controls tumor growth of hormone insensitive tumors and provides a bidirectional effort to prevent autocrine stimulated growth in hormone insensitive tumors of prostate and breast origin. In addition, the targeted mRNAs have sequence homology to additional oncogenes, whose expression is either elevated or prognostically significant in tumors. Therefore, these two agents may simultaneously inhibit expression of two arms of a single growth regulatory pathway, and in addition inhibit several homologous oncoproteins important for progression of malignancies. Initially directed against tumors of the prostate and breast, these compounds are now being evaluated for their efficacy against glioblastomas of the brain. These antisense oligos consist of relative short strands of synthetic DNA bases complementary in sequence to the mRNAs encoding TGF-α and EGFR. When bound to the mRNA via hybridization, the mRNA is though to be destroyed by RNAse activity, leading to a decrease in the targeted protein's translational expression. TGF- α and EGFR are thought to have roles in prostate cancer growth and EGFR is homologous to erbB-2, which is prognostically significant in breast cancers. EGFR is also over expressed and growth regulatory in glioblastomas. We have generated data demonstrating both in vitro and in vivo growth inhibitory effects of these agents against all three types of tumors, as well as their cellular uptake using delivery vehicles. The Novartis Corporation has provided us with additional oligos formulated to include our patented nucleotide base sequences, but having a second generation DNA backbone. We find that in oligos which share the same base sequence, differences in the backbone can have dramatic effects, either in enhancing activity or in its inhibition, when tested against different cell lines.

Development of new methods to enhance the specific delivery of therapeutic agents.

For in vitro evaluation of drug efficacy it is important that the agent enter its target. To do this we have labeled antisense oligos with a fluorescent tag to observe their intracellular uptake and localization. Experiments have indicated that for in vitro studies to identify effective oligos, entry was enhanced by the use of the cationic lipid, lipofectin and the use of this agent is necessary for new oligo effectiveness to be evaluated. The use of lipofectin, however, presents other questions. Several of the delivery mechanisms employed for specific targeting requires that the oligos be biotinylated. Until our study, no evaluation as to the effect of lipofectin on the uptake of biotinylated agents had been done.

In vivo delivery presents two additional challenges. The first is to design specific delivery systems which direct oligos solely to tumor targets while sparing normal adjacent tissue. The second is the identification of appropriate targets for the oligo to be specifically directed towards. These in vivo questions were addressed by the construction of two types of delivery vehicles which were evaluated for the specific delivery of radioactively labeled oligos (or potentially other chemotherapeutic agents) to prostate cancer cells expressing either the well characterized prostate specific antigen (PSA) or the more recently studied prostate specific membrane antigen (PSMA). These antigens differ in their level and site of expression, their amount of shedding into the circulation and also by monoclonal antibody recognition and binding.

The first delivery vehicle constructed was a hybrid monoclonal (also known as a heteroconjugate) which recognizes both PSA expressed by prostate tumors and biotin, a hapten-like conjugate linked to the oligo sequence. The second delivery vehicle type was a modification of monoclonal antibodies specific for either PSA or PSMA. The anti PSA antibody is commercially available, however the anti PSMA directed monoclonal antibody (CYT 356) was the provided by the Cytogen Corporation. Each monoclonal was modified by conjugating avidin to those carbohydrates associated with the Fc portion of the respective antibody. Therefore, these monoclonals recognize PSA or PSMA on prostate cancer cells as well as the biotin conjugated to the oligo. We have demonstrated, using mobility shift assays, that both types of delivery vehicles associate with the biotinylated oligos, and are able to direct 35S labeled oligos to prostate tumor cells. In vitro, cellular uptake of oligos following delivery with either vehicle type has been visualized by FITC labeled biotinylated oligos. Our data demonstrate that the second type of delivery vehicle, which uses conjugated avidin in the Fc region, is more effective and efficient in delivering oligo to tumors regardless of whether PSA or PSMA is targeted. This is due to the ability of avidin to bind multiple biotinylated oligos and also to the higher efficiency in the preparation of this type of antibody. We also find that PSMA is a better prostate tumor target than PSA because PSMA is internalized in prostate cancer cells, whereas PSA is shed into the serum. Therefore, greater amounts of radio labeled oligo remains in serum when either delivery vehicle type specifies PSA. In contrast, more label is recovered from prostate tissue when PSMA is targeted by delivery vehicles.

Development of combination therapy in the prostate cancer models.

Antisense oligo are also being administered in the laboratory in combination with traditional chemotherapeutic agents, such as taxol. These studies may identify whether certain oligo/chemotherapy agent combinations can enhance efficacy in a synergistic manner, or identify those which act additively. The combination of relatively non-toxic oligos with traditional chemotherapy drugs may diminish the toxicity associated with the later agents.

Evaluation of antisense therapy on gene expression using microarray assay.

In a collaborative effort with the Brain Tumor Research Program at Children's Memorial Hospital, we have used microarray analysis to evaluate the mechanism of antisense activity. In an abstract accepted for presentation at the 2002 AACR meeting we compare the relative amounts of mRNA present following the treatment of prostate cancer cell lines with antisense directed against the epidermal growth factor receptor (EGFR). This protein is important in glioblastoma development and therefore of interest to this brain tumor research group. We find that the mechanisms for antisense activity in prostate cancer cells are complex. Cells incubated with antisense oligos directed against EGFR, at a concentration which is known to inhibit cell growth, as well as to diminish the absolute amount of EGFR protein, have levels of encoding mRNA not significantly different from cells similarly treated with control oligos having a scrambled base sequence.

Urologic Oncology

The various SEER data bases, provided by the National Cancer Institute have been evaluated for the equity of care being provided between members of different racial groups who have prostate cancer. Using this data base the incidence of cancer development and the frequency of treatment potentially able to cure can be evaluate. Therefore the aggressiveness of cancer therapy as applied to different groups can be evaluated.

Patents:

UNITED STATES PATENT #5,891,858 issued April 6, 1999.
Title: Antisense Polynucleotide Inhibition of Epidermal Human Growth Factor Receptor Expression. Inventor: Marvin Rubenstein, Skokie, IL Assignee: Hektoen Institute for Medical Research

UNITED STATES PATENT #5,610,288 issued March 11, 1997.
Title: Antisense Polynucleotide Inhibition of Epidermal Human Growth Factor Sensitive Cells. Inventor: Marvin Rubenstein, Skokie, IL Assignee: Hektoen Institute for Medical Research