Adam P. Dicker

Dihydrofolate reductase as a therapeutic target.

The folate antagonists are an important class of therapeutic compounds, as evidenced by their use as antiinfective, antineoplastic, and antiinflammatory drugs. Thus far, all of the clinically useful drugs of this class have been inhibitors of dihydrofolate reductase (DHFR), a key enzyme in the synthesis of thymidylate, and therefore, of DNA. The basis of the antiinfective selectivity of these compounds is clear; the antifolates trimethoprim and pyrimethamine are potent inhibitors of bacterial and protozoal DHFRs, respectively, but are only weak inhibitors of mammalian DHFRs. These species‐selective agents apparently exploit the differences in the active site regions of the parasite and host enzymes. Methotrexate is the DHFR inhibitor used most often in a clinical setting as an anticancer drug and as an antiinflammatory and immunosuppressive agent. Considerable progress has been made recently in understanding the biochemical basis for the selectivity of this drug and the biochemical mechanism (or mechanisms) responsible for the development of resistance to treatment with the drug. This understanding has led to a new generation of DHFR inhibitors that are now in clinical trials.— Schweitzer, B. I.; Dicker, A. D.; Bertino, J. R. Dihydrofolate reductase as a therapeutic target. FASEB J. 4: 2441‐2452; 1990.

The effect of treatment positioning on normal tissue dose in patients with prostate cancer treated with three-dimensional conformal radiotherapy

PURPOSE To prospectively assess the effect of supine vs. prone treatment position on the dose to normal tissues in prostate cancer patients treated with the three-dimensional conformal technique. METHODS AND MATERIALS Twenty-six patients underwent three-dimensional treatment planning in both the supine and prone treatment positions. The planning target volume and normal tissue structures were outlined on each CAT scan slice, and treatment plans were compared to assess the effect of treatment position on the volume of rectum, bladder, and bowel exposed to the high dose of irradiation. RESULTS The average dose to the rectal wall and the V95 (volume of rectal wall receiving at least 95% of the prescription dose) for the prone position were 64 and 24% of the prescription dose, respectively, compared to 72 and 29%, respectively, for the supine position (p < 0.05). When the average rectal wall dose was used as an endpoint, 14 of the 26 patients (54%) had an advantage for the prone position compared to 1 (4%) who demonstrated an advantage for the supine position (p < 0.0002). Similarly, when V95 of the rectal wall was used as a measure of comparison, 15 patients (58%) had an advantage for the prone position compared to 1 (4%) who demonstrated an advantage for the supine position (p < 0.0002). In 13 patients (50%), a change from supine to the prone position was associated with reduction of the V95 to levels < 30% of the prescription dose compared to 3 patients (11%) in whom such an advantage resulted from change of the prone to the supine position (p < 0.005). The effect of treatment position on the rectal wall dose was most pronounced in the region of the seminal vesicles. An increased volume of bowel was also noted in the supine position. The treatment position, however, had no significant impact on the dose to the bladder wall. CONCLUSIONS Three-dimensional conformal radiotherapy for prostate cancer in the prone position is associated with significant reduction of the dose to the rectum and bowel resulting in an improvement in the therapeutic ratio.

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi‐drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium‐dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r‐sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r‐sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non‐ionic detergent Triton X‐114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium‐dependent translocation of sorcin in the specialized functions of sorcin‐expressing cells is discussed.

Quantitation of gene copy number and mRNA using the polymerase chain reaction.

Quantitation of the copy number of a specific gene in a given sample on the genomic as well as on the steady state RNA transcriptional level is of great interest in both basic and clinical research. Traditionally, the copy number of a gene in a given sample is estimated by Southern blot or dot blot hybridization techniques. A certain amount of total genomic DNA of a sample is digested with restriction enzymes, size fractionated by nondenaturing agarose gel electrophoresis, and transferred and permanently bound to a nitrocellulose or nylon membrane (Southern blot); alternatively, DNA can be transferred and bound to a membrane without previous manipulations (dot blot). The membrane is then incubated with a radioactive, labeled molecular probe, which specifically binds to the gene of interest. After washing steps to remove unbound probe, the membrane is exposed to film and a signal is detected. The intensity of the signal, which can be quantitated (e.g., by densitometry), correlates to the amount of specific DNA (gene of interest) per amount of total genomic DNA and gives an estimate of the copy number of this gene. Similarly, the degree of expression of a gene can be estimated after transferring a certain amount of RNA to a membrane (Northern blot) and hybridization to a specific probe. In each experiment, control samples with a known degree of gene expression have to be analyzed in parallel. However, there are several limitations of the above techniques. First, the sensitivity of these assays allows only for detection of gross differences in copy number and expression of a certain gene. Usually, only differences between samples of more than 3- to 4-fold can be detected. Moreover, very low levels of expression of a gene of interest may not be detectable at all.

Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue

One of the greatest potentials of polymerase chain reaction (PCR) lies in the fact that even minute amounts of target DNA or extensively damaged DNA can be successfully amplified in vitro and thus become amenable to further study. This enables a detailed molecular analysis of small amounts of DNA from tissue that has been damaged by fixation (e.g., in formalin) and long-term storage in paraffin. The applications of this methodology are nearly unlimited. For example, rare tumors that are stored as formalin-fixed, paraffin-embedded tissue in pathology departments throughout the world can be analyzed at the molecular level. Furthermore, tissue from small lesions (e.g., primary skin melanomas), which are only rarely available for molecular analysis since the entire specimen is usually needed for histopathological assessment, can be examined. For PCR analysis, only several sections from the paraffin block, which are usually dispensable, are sufficient. Even small amounts of very low quality DNA can be used, as the sensitivity of the detection of specific target DNA sequences is several orders of magnitude higher than that with any conventional method. For example, Southern blot analysis of DNA from paraffin-embedded tissue has been performed, but with limited success as only relatively small amounts of degraded and irreversibly modified DNA can be obtained from embedded specimens (1,2). Thus, PCR methodology creates an ideal link between traditional histology and modern molecular biology (3).

Effect of Codon 22 Mutations on Substrate and Inhibitor Binding for Human Dihydrofolate Reductase

Previous studies in this laboratory have identified an altered dihydrofolate reductase (DHFR) enzyme in the methotrexate (MTX) resistant Chinese hamster ovary (CHO) cell line Pro-3MtxRIII [1,2]. The alteration involved the replacement of the active site residue Leu (L, position 22) by Phe as the result of a base transition at nucleotide 67 in the DHFR gene [3]. Enzyme kinetic studies of the altered enzyme revealed that the Km for both substrates (H2folate & NADPH) were similar when compared with wild-type vertebrate enzyme, but the Ki for MTX was increased some 100-fold. Interestingly, previous studies by other researchers have also identified a mutation at residue 22 in MTX-resistant cell lines: a Leu to Arg mutation in the mouse line 3T6-R400 [4], and a Leu to Phe mutation in yet another CHO cell line [5]. Combined, these results suggest that codon 22 may be a “hot spot” which readily mutates to impart a MTX-resistant phenotype to its host cell. While definitive proof still awaits, it seems plausible that these or similar mutations in DHFR may be responsible for some instances of resistance in tumors of patients who receive antifolates therapeutically.

Manual and automated direct sequencing of product generated by the polymerase chain reaction.

Identification of point mutations has been facilitated by a number of techniques, including transfection assays, oligonucleotide hybridization, electrophoretic migration of heteroduplexes, RNase mismatch analysis, direct sequencing, and DNA-polymerase catalyzed amplification. The large number of available techniques emphasizes the importance of developing rapid and reliable methods to identify molecular changes in genes. To date, we have concentrated on exploiting DNA-polymerase catalyzed amplification methods (1,2) in conjunction with direct manual and automated DNA sequencing to detect point mutations in the dihydrofolate reductase (DHFR) gene of methotrexate-resistant cells.