Biomedical subjects
Publications and source records attributed to R Seshadri.
A recently described retinoblastoma cell line, FMC-RB1, showed a 16-fold N-myc oncogene amplification. The patient from whom the cell line was obtained died from an aggressive disease. It is suggested that N-myc amplification may be an adverse prognostic indicator.
A mass spectral characterization of a set of anthracyclines and anthracyclinones comprised of daunorubicin, adriamycin and their modified analogs was carried out by using negative and positive fast atom bombardment (FAB) ionization techniques. Addition of more than one hydrogen to the molecular ions of the anthracyclines was observed. The choice of the FAB matrix played an important role in the characterization of these compounds. The dominant ions in the molecular ion region were M-. (or M+.) and MH- (or MH+.2) when sulfolane and glycerol, respectively, were employed as the FAB solvents. The major fragmentation was cleavage of the glycosidic bond with the charge retention mainly on the aglycone moiety. Aromatization of the tetracyclic ring promoted further fragmentation of the aglycone moiety. The anthracyclinones could be characterized only by negative FAB ionization using sulfolane as the FAB matrix. The assigned fragmentation pathways were confirmed by acquiring metastable ion spectra using B/E linked-field scans.
A patient-motivated pre-operative autologous blood transfusion service is described. The programme is organized and staffed by members of the Transfusion Service. In this programme patients who are enrolled for preoperative autologous blood collection are selected from elective surgical waiting lists without a formal referral from the surgeon. Since starting the scheme the average number of units donated per month has increased from 8 to 33 and complications have been minor. The system has proved to be practical and cost-effective, and it has a definite place as part of an overall approach to the prevention of transfusion-related complications.
The rare occurrence of Wiskott-Aldrich syndrome (WAS) in identical twin brothers without a preceding family history of the disease is described. Investigation into the nature of the lymphocyte abnormality in the surviving twin suggests that the frequency of T cell clone forming cells is markedly decreased. Further analysis of the T cell clones showed a deficiency of CD8 positive cytotoxic/suppressor cells. This alteration may be an underlying mechanism in the development of some of the clinical manifestations of WAS.
Both growth factors and steroid hormones are known to be associated with breast cancer. We have studied the epidermal growth factor receptor (EGFR) in canine mammary tumors and have shown the presence of a single class of 125I-EGF binding receptor sites in 9 out of 13 (70%) tumors, using Scatchard plot. The dissociation constant (KD) was 10(-9)M. An inverse correlation between EGFR and estrogen receptor (ER) was observed in 54% of the tumors. Our data suggest that EGFR might be used as a biochemical marker in canine mammary tumors.
The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.
Cytotoxic drug sensitivity of normal human lymphocytes and malignant lymphocytes was estimated by a clonogenic method. Malignant T and B lymphocytes were relatively more sensitive than normal T lymphocytes to vincristine and Adriamycin. Since no plateau was observed in the clone survival with associated increasing drug concentration, spontaneous mutants could not be detected. It is suggested that the resistance to "natural product" drugs such as vincristine arises from induced mutations and not from the selection of already existing spontaneous mutants.
Multiple drug resistance has been shown to be associated with amplification/increased expression of a gene designated MDR. The localization of one member of the MDR gene family, MDR1, to the long arm of chromosome 7 by in situ hybridization is reported.
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Gene rearrangements of immunoglobulin and T cell antigen receptor gene loci were studied in 67 patients referred to a general hematology clinic. The results of gene analysis supported the clinical diagnosis in most cases where involved tissue was studied. However, in a number of cases, gene rearrangement studies gave unexpected results of possible diagnostic significance. Thus two patients who presented diagnostic difficulties were finally diagnosed as having non-Hodgkin's lymphoma; gene analysis suggested that the initial diagnosis of Hodgkin's disease may have been correct. In two patients who had chronic lymphocytosis and were suspected of having chronic lymphocytic leukemia, no evidence of monoclonal disease of B or T cells could be found and the basis for the lymphocytosis was presumed to be non-malignant. Gene analysis was able to detect monoclonal B cell disease in the otherwise normal blood of two of six patients with lymphoma, one of two patients with macroglobulinemia, and none of six patients with myeloma. The study of gene rearrangements may be practical aid to diagnosis in some situations of uncertainty.
Pretransfusion testing has undergone a rationalization, resulting in a more efficient transfusion service but with no compromise to patient safety. Current debate centres on the necessity of performing an antiglobulin crossmatch if a suitable screening test for detecting red cell antibodies is negative. A retrospective study of all pretransfusion testing was undertaken to determine the specificities and possible clinical significance of antibodies detected by the crossmatch alone. The results indicate that omission of the crossmatch will not compromise patient safety. This will have benefits to both the patient and the laboratory. The patient will benefit because of less time needed to provide homologous donor blood. The laboratory will benefit from cost savings as a result of less antiglobulin reagent being used, less time taken for providing blood (and hence further cost savings) and less blood needing to be held in stock. The cost savings generated could be channelled into areas that will result in greater patient safety at a lower cost than crossmatching. Two of these areas are prevention of patient and specimen identification errors by the use of a unique transfusion number system and the full utilization of an autologous blood service.
The N-acylanthracyclines AD32 (N-trifluoroacetyladriamycin-14-valerate) and AD143 (N-trifluoroacetyladriamycin-14-O-hemiadipate) are analogs of Adriamycin (ADR) undergoing clinical or advanced pre-clinical screening. Their principal metabolites, following the cleavage of the 14-acyl side-chain, are N-trifluoroacetyladriamycin (AD41) and its reduced form N-trifluoroacetyladriamycinol (AD92). Both these compounds are biologically active and detectable in treated patients, laboratory animals, and in tissue culture cells. Unlike ADR, AD32, as well as AD143 and metabolites, show no detectable binding to double-strand DNA. Their effects on DNA have been previously investigated in vivo and in vitro using the alkaline filter-elution assay. It has been shown that all of the compounds cause approximately equivalent amounts of protein-associated DNA breaks (PAB) and DNA-protein crosslinks in a mouse lymphoma and in tissue-culture leukemia cells. In order to establish whether the induction of PAB by the drugs requires DNA topoisomerase II mediation, cleavage mapping analysis was done with tested compounds using purified human topoisomerase II. DNA fragmentation was significantly enhanced in the presence of the enzyme and either AD41 or AD92. In contrast, no fragmentation enhancement was observed in the presence of the parental drugs AD32 or AD143. The results strongly suggest that metabolic activation of N-acylanthracyclines by nonspecific esterases is a prerequisite for their interaction with DNA topoisomerase II and for stabilization of the cleavable complex.
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A new continuous cell line derived from an untreated human retinoblastoma has been established. This cell line, FMC-RB1 is strongly positive for common acute lymphoblastic leukemia antigen and shows a number of ring chromosomes and two marker chromosomes considered to be derivations of chromosome #17; the nonrandom chromosomal changes associated with retinoblastoma, particularly the loss of a chromosome #13 or the deletion of 13q14 was not observed. The establishment of the cell line initially required the presence of bone marrow stromal cells. Morphologically, this cell line grew as a suspension of small round cells in grape-like clusters with periodic "shedding" of single cells. FMC-RB1 could be cloned in soft agar, even in the absence of bone marrow stromal cells as "feeders", making it suitable for a variety of biological studies.
A cell line (RCH-ACV) was established from a bone marrow sample of a child with acute lymphoblastic leukemia (ALL). The cell line lacked Epstein-Barr virus nuclear antigen and exhibited a recently described nonrandom chromosome translocation, 1;19, thought to be associated with pre-B-ALL and poor prognosis. Banding studies confirm that the breakpoint of chromosome #19 occurs at p13.3. Cell surface marker analysis using a panel of monoclonal antibodies revealed markers consistent with common ALL phenotype. Although the cells did not show cytoplasmic immunoglobulin, studies of the immunoglobulin gene rearrangement confirmed the pre-B phenotype. This cell line could be of great value to studies of the role of the specific translocation 1;19 in the etiology of pre-B-ALL.
On the consideration that the highly active DNA-nonbinding adriamycin analogues N-(trifluoroacetyl)adriamycin 14-valerate and N-(trifluoroacetyl)adriamycin 14-O-hemiadipate undergo initial metabolic conversion to N-(trifluoroacetyl)adriamycin by the action of nonspecific serum and tissue esterases, a number of N-(trifluoroacetyl)adriamycin 14-thio esters have been prepared and studied for in vitro growth inhibition, vs. human-derived CCRF-CEM leukemic lymphocytes, and in vivo antitumor activity, vs. murine P388 leukemia, relative to the rate of thio ester deacylation induced by esterases present in mouse serum. Products were obtained by reaction of N-(trifluoroacetyl)-14-bromodaunorubicin with thioacetic, thiopropionic, thiobutyric, thiovaleric, and thiobenzoic acids in ethanol, in the presence of potassium carbonate. Because little is known about similar thio ester derivatives of adriamycin itself, the corresponding adriamycin 14-thio esters were also prepared and evaluated for antitumor activity; with these products, determination of their extent of interaction with calf thymus DNA was also performed. For the adriamycin thio ester products, significant in vivo anti-P388 activity was seen with the thioacetate, thiovalerate, and thiobenzoate derivatives, although no compound matched the curative effects of N-(trifluoroacetyl)adriamycin 14-valerate in this system. With respect to the N-(trifluoroacetyl)adriamycin 14-thio ester products, although the corresponding oxo ester analogues are all significantly biologically active, none of the thio ester derivatives showed activity in vitro or in vivo.
In connection with structure-activity studies related to the novel DNA-nonbinding adriamycin analogues N-(trifluoroacetyl)adriamycin 14-valerate (AD 32) and N-(trifluoroacetyl)adriamycin 14-O-hemiadipate (AD 143), we have now prepared a series of N-(trifluoroacetyl)adriamycin derivatives with N-acylamino acid esters at the 14-carbinol position. Target compounds were made by reaction of N-(trifluoroacetyl)-14-iododaunorubicin with the sodium salts of N-acylamino acids generally in dimethylformamide-ethylene glycol solvent. Products were evaluated for in vitro growth-inhibitory activity and, to a limited extent, in vivo antitumor activity in the murine P388 leukemia system. ID50 values for the target compounds vs. cultured CCRF-CEM cells were generally in the same range as those for the above-mentioned DNA nonbinding adriamycin analogues. Of the four compounds tested for in vivo activity, although none was as effective as N-(trifluoroacetyl)adriamycin 14-valerate, all showed significant activity in the P388 assay system, with three of the compounds, at the doses used, being essentially equiactive with an optimal dose of adriamycin. Studies on the rate of esterase-mediated deacylation of the products, in a defined system containing unfractionated mouse serum as the source of enzyme, showed no relationship between the in vitro and in vivo activities of these compounds and the relative ease at which the side-chain ester substituents were hydrolyzed.