Biomedical subjects

T C Hamilton

Publications and source records attributed to T C Hamilton.

At least 73 records · Page 4Linked to original sources

PubMed1994

Spontaneous transformation of rat ovarian surface epithelial cells results in well to poorly differentiated tumors with a parallel range of cytogenetic complexity.

We previously used rat ovarian surface epithelial cells subjected to repetitious growth in vitro to provide experimental evidence in support of a role for incessant ovulation in the etiology of ovarian cancer. We have now initiated a series of 30 independent rat ovarian surface epithelial cell lines. This report describes findings in eight of the cell lines that, based on tumor formation in athymic mice, have undergone malignant transformation. Each of the tumors exhibited chromosomal alterations. Two well to moderately differential tumors had only one or two cytogenetic changes, and they had in common the presence of numerical gains. Each of five poorly differentiated tumors had complex karyotypes with three to eight clonal aberrations, prominent among them being unbalanced rearrangements and numerical losses. Several poorly differentiated tumors also had marker chromosomes, double minutes, or homogeneously staining regions. These findings demonstrate that the malignant tumors produced by spontaneously transformed rat ovarian surface epithelial cell lines range in degree of differentiation, which is paralleled by the cytogenetic complexity. Thus, this model system may fill an important void in future efforts to define the genetic basis of common epithelial tumors of the ovary and many features characteristic of these neoplasms.

Adenocarcinoma↗

PubMed1994

K+ channel activators, acute glucose tolerance and glibenclamide-induced hypoglycaemia in the hypertensive rat.

The effects of the K+ channel activators, levcromakalim, pinacidil and diazoxide, at comparable antihypertensive doses, on acute glucose tolerance and glibenclamide-induced hypoglycaemia were examined in conscious spontaneously hypertensive rats (SHR). Levcromakalim (0.15 mg.kg-1 p.o.) and pinacidil (1.0 mg.kg-1 p.o.) caused a slight, but short-lived, impairment of glucose tolerance following oral or s.c. administration of glucose (2.0 g.kg-1). This effect, although small, was abolished by the beta-adrenoceptor blocker, propranolol (2.0 mg.kg-1 p.o.). Diazoxide (30.0 mg.kg-1 p.o.) caused a marked and sustained impairment of oral glucose tolerance and s.c. glucose tolerance, the profile of which was quantitatively and qualitatively different from levcromakalim or pinacidil and was not significantly affected by propranolol. Glibenclamide (1.0-10. mg.kg-1 p.o.) elicited a dose-related hypoglycaemic response. Levcromakalim or pinacidil had little or no significant effect on the hypoglycaemic response elicited by glibenclamide (3.0 mg.kg-1). Conversely, diazoxide both abolished and reversed glibenclamide-induced hypoglycaemia. We conclude that levcromakalim and pinacidil have only marginal and transient effects on glycaemic control in conscious SHR and that these disturbances are probably mediated indirectly via reflex activation of the sympathetic nervous system in response to blood pressure lowering. In addition, at active antihypertensive doses neither levcromakalim nor pinacidil significantly interfered with the ability of glibenclamide to reduce blood glucose concentration. Diazoxide's impairment of oral glucose tolerance, s.c. glucose tolerance and glibenclamide response confirms this drug's well known ability to activate pancreatic KATP channels.

Administration, Oral↗

PubMed1994

Role of platinum-DNA adduct formation and removal in cisplatin resistance in human ovarian cancer cell lines.

A series of cisplatin-resistant cell lines were used to examine the formation and removal of platinum-DNA adducts from the overall genome and the formation and removal of cisplatin-interstrand cross-links from specific genomic regions. Cisplatin accumulation and DNA platination levels, which correlated linearly, were similar in three of the resistant cell lines despite differences in their primary cisplatin resistance. Increased platinum removal from total genomic DNA was found to be associated with increased resistance. Interstrand cross-link levels were found to be 2- to 4-fold lower in the 28S ribosomal RNA gene and a non-coding genomic region of the resistant cell lines as compared with the parental A2780 cell line. In addition, 1.2- to 2.7-fold more cross-links were formed in the non-coding region than in the ribosomal RNA gene in all of the cell lines. Interstrand cross-links were removed more rapidly from both regions of the highly cisplatin-resistant C80 and C200 cells and from the ribosomal RNA gene only in the cell lines of lower resistance. The results support a role for DNA repair and alterations in interstrand cross-link formation in cisplatin resistance and provide evidence for heterogeneous interstrand cross-link formation in the genome.

Cisplatin↗

PubMed1994

Phase I and pharmacokinetic study of ormaplatin (tetraplatin, NSC 363812) administered on a day 1 and day 8 schedule.

Ormaplatin (tetraplatin, NSC 363812) is a platinum(IV) analogue that is active against cisplatin-resistant cell lines in preclinical models. A schedule previously shown to be active and well tolerated for cisplatin was evaluated in 26 patients. Ormaplatin was administered over a dose range of 4.4-60.8 mg/m2 i.v. given over 30 min on a day 1 and day 8 schedule every 28 days. Twenty-three patients had received prior chemotherapy, and the median performance status was 1. Nausea/vomiting (> or = grade 2) occurred in 40% of patients but was well controlled with standard antiemetic therapy. One patient had grade 2 renal toxicity and 1 patient had grade 3 hepatotoxicity (grade 2 pretreatment). No toxicity limited the dose given during the first course. With repeated drug administration delayed severe neurotoxicity developed in 4 patients, manifested as a sensory polyneuropathy in 3 patients and a possible autonomic neuropathy in one. Prospective nerve conduction studies did not detect subclinical neuropathy prior to the onset of symptoms. Patients who received cumulative doses above 200 mg/m2 were at increased risk for developing neurotoxicity. Plasma elimination of ultrafilterable platinum (measured by atomic absorption spectrometry) was biphasic with a harmonic mean terminal half-life of 15.8 h. The mean total body clearance and renal clearance of ultrafilterable platinum were 173 and 29.8 ml/min/m2, respectively. Thus, renal clearance accounted for 16% of total clearance suggesting that extensive protein/tissue binding was responsible for the majority of platinum clearance. Approximately 60% of the platinum is protein bound (one-half irreversibly) at the end of the infusion. Pharmacokinetic parameters were not dose dependent. No pharmacokinetic parameters were more predictive of neurotoxicity than the cumulative ormaplatin dose. A phase II dose cannot be recommended on this schedule because severe and unpredictable neurotoxicity precludes the administration of more than three cycles at the three highest doses levels tested.

Adult↗

PubMed1994

Antitumor activity and biochemical effects of aphidicolin glycinate (NSC 303812) alone and in combination with cisplatin in vivo.

Aphidicolin, an inhibitor of DNA polymerases alpha and delta, is cytotoxic in vitro against tumor cells. The poor solubility of aphidicolin has led to the development of aphidicolin glycinate (AG; NSC 303812), a water soluble ester currently in early clinical trials. The antitumor activity of AG was investigated in a series of transplantable murine tumors in vivo. The drug demonstrated activity against the i.p. implanted B16 melanoma, producing maximum increased life spans of 75% following i.p. administration every 3 h for three doses on days 1-9. Treatment schedules involving both single injections per day on days 1-9 and multiple injections per day on days 1, 5, and 9 were less effective, indicating that this antitumor activity is schedule dependent. Similarly, greater activity was observed against the i.p. M5076 sarcoma when three daily injections were given on days 1-9 (57% increased life span) than with a single injection either on days 1-9 (36% increased life span) or on days 1, 5, 9, and 13 (inactive). Further scheduling studies in the s.c. M5076 sarcoma model showed that a 7-day infusion was superior to both a 24-h infusion and a 7-day course of three bolus treatments per day. On the assumption that DNA polymerase inhibition is the basis for this antitumor activity, inhibition of DNA synthesis in BALB/c x DBA/2 F1 mice was investigated by measuring incorporation of [3H]thymidine (20 microCi, i.v.) into DNA of spleen and jejunum. At 2 h after administration of AG, inhibition of DNA synthesis was dose dependent (median inhibitory dose, 60 mg/kg in both tissues) and was > 99% at 300 mg/kg. The inhibition was rapid in onset; AG (100 mg/kg i.p.) produced maximal (> 98%) inhibition in both tissues at 30 min. Recovery occurred in the intestine within 16 h; in spleen recovery was delayed to 24 h, and was followed by a rebound incorporation at 48 h (203%). A comparison of the inhibition of thymidine incorporation in tumor cells (B16 melanoma and P388 leukemia) and normal jejunum revealed no significant differences in the extent of inhibition or the rapidity of recovery in these tissues. The rapid recovery of DNA synthesis inhibition supports the use of prolonged infusion schedules in clinical trials, but the lack of evidence of selectivity for tumor cells suggests that AG may be of limited therapeutic value as a single agent. Thus, we evaluated AG in combination with cisplatin in an in vivo model of cisplatin refractory human ovarian cancer.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Transcriptional activation of c-myc proto-oncogene by estrogen in human ovarian cancer cells.

NIH:OVCAR-3 is a human ovarian cancer cell line that expresses a moderate amount of estrogen receptors, 28 fmol/mg protein. We have found that estrogen at a concentration of 10(-7) M induced a 2.3-fold increase in the growth of NIH:OVCAR-3 cells after 48 h stimulation. A 4-fold increase in c-myc mRNA expression at 30 min and a 7.5-fold increase at 50 min post-induction with estradiol were observed. Nuclear run-on analysis indicated that c-myc transcripts increased 4-fold within 10 min of estrogen addition. The half-life of c-myc mRNA was 64 min +/- 5 min and was not affected by estrogen. Antisense oligonucleotide to c-myc specifically inhibited the estrogen stimulated c-myc protein expression as well as the growth of NIH:OVCAR-3 cells. A control ovarian cancer cell line OC-3-VGH that had few estrogen receptors (1 fmol/mg protein) did not respond to estrogen in growth; however, these cells respond to estrogen with a 1.5-fold increase in c-myc mRNA. The stability of c-myc mRNA of these cells was not affected by estrogen. Our results indicate that transcriptional induction of c-myc expression by estrogen plays a critical role in the proliferation of NIH:OVCAR-3 cells.

Cell Division↗

PubMed1993

Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines.

We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to > 400-fold). These cells had strikingly increased (4- to 50-fold) levels of glutathione (GSH) as compared with the drug-sensitive cells of origin (A. K. Godwin et al., Proc. Natl. Acad. Sci. USA, 89: 3070-3074, 1992). Utilizing this panel of resistant cell lines, we evaluated cross-resistance to classical alkylating agents, natural product drugs, and irradiation. We observed that cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance. Similarly, melphalan cross-resistance continued to increase to > 400-fold and again paralleled the primary cisplatin resistance. Cell lines with low to very high levels of resistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxin derivative etoposide. Cross-resistance is also observed for other natural product drugs, including Adriamycin (approximately 80-fold), mitoxantrone (approximately 440-fold), and taxol (approximately 40-fold). Cross-resistance to irradiation is, however, modest (< 2-fold). The cells with the greatest primary resistance to cisplatin most commonly had the highest cross-resistance to the other drugs examined. The cross-resistance to the natural product category drugs was found not to be mediated by the products of either the multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes based on lack of coordinate increased expression or amplification of these genes as assessed by Northern and Southern blot analyses. Furthermore, verapamil failed to markedly increase drug sensitivity. Although there was no indication that these natural product drug efflux pumps were operative, we observed decreased doxorubicin accumulation in these cell lines cross-resistant to natural products. In addition, alternations in DNA topoisomerase II mRNA levels, which have been observed in a variety of human tumor cell lines selected in vitro for resistance to etoposide or teniposide, were not detected. Only intracellular levels of GSH correlated with cross-resistance to these diverse anticancer agents and partial loss of resistance was associated with a marked decrease in glutathione levels. In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least in part, be the direct result of GSH-mediated drug inactivation or may be due to a combination of GSH conjugation to drug and conjugate efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Variable baseline gamma-glutamylcysteine synthetase messenger RNA expression in peripheral mononuclear cells of cancer patients, and its induction by buthionine sulfoximine treatment.

The role of glutathione (GSH) in tumor cell resistance to alkylating agents and platinum compounds is suggested by a body of laboratory and clinical studies. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS), the expression of which is proportional both to GSH content and to the level of resistance in ovarian cancer cell lines. The role of this enzyme in regulating GSH levels is unclear, however. Reversal of resistance is achieved in vitro and in vivo with the use of buthionine sulfoximine (BSO), a potent inhibitor of gamma-GCS. In the course of a Phase I clinical trial of BSO and melphalan, we have measured GSH and expression of gamma-GCS mRNA in peripheral mononuclear cells before and at intervals after the initiation of treatment with BSO. Mean baseline GSH content was 6.89 nmol/mg protein. Treatment with BSO (10.5 to 17 g/m2 i.v. every 12 h for six doses) resulted in a mean nadir GSH decline to 19% of control values, most commonly on day 3. Baseline expression of gamma-GCS mRNA was measured by a reverse transcriptase polymerase chain reaction-based method. When described relative to that of beta-actin, the expression of gamma-GCS varied over 3-fold among individuals. Following GSH depletion by BSO, the level of gamma-GCS mRNA rose successively on days 3 and 5 to reach a mean increase of 2-fold on day 8. Differences were observed among patients in their capacity to respond to GSH depletion by increasing gamma-GCS steady-state mRNA levels (1.4- to 3.1-fold). These results show that the expression of gamma-GCS is variable in the population and suggest that the cellular content of GSH may be involved in the regulation of its expression.

Adult↗

PubMed1993

Transformation of rat ovarian epithelial and Rat-1 fibroblast cell lines by RAST24 does not influence cisplatin sensitivity.

Recent reports suggest that expression of an activated c-Ha-ras oncogene is associated with cisplatin resistance in NIH-3T3 fibroblasts. To investigate the generality of these observations, cisplatin cytotoxicity was determined in a series of clonal Rat-1 fibroblast and rat ovarian surface epithelial (ROSE) cell lines carrying a zinc-inducible metallothionein-RAST24 fusion gene, MTRAST24. Cisplatin sensitivity in RAS-transformed fibroblast sublines did not differ from parental controls. Induction of mutant RAST24 expression by zinc sulfate did not affect the cisplatin sensitivity of individual cell lines. Expression of mutant p21Ha-RAS varied more than 40-fold in these fibroblast sublines. Similarly, there was no difference in cisplatin sensitivity between parental ROSE controls, neomycin phosphotransferase transfected controls, or MTRAST24 transfectants. Finally, the cisplatin sensitivity of RAS-transformed ROSE cells was similar to that of spontaneously transformed ROSE cells. Overall, these observations suggest that there is little relationship between mutant ras expression and cisplatin sensitivity in rat epithelial and fibroblast cell lines.

Animals↗

PubMed1993

Mechanisms and modulation of resistance to chemotherapy in ovarian cancer.

Chemotherapy for advanced ovarian cancer remains suboptimal. Despite the improvements in objective response rates realized with cisplatin-based combination chemotherapeutic regimens, most patients still die of refractory cancer. Drug resistance has emerged as the single most important determinant of treatment outcome. Laboratory studies have provided substantial insights into the cellular mechanisms of resistance to the commonly used chemotherapeutic agents. Decreased drug accumulation, metabolic drug inactivation, and repair or tolerance to drug-induced cellular injury all contribute to resistance at the cellular level. Identification of these mechanisms has facilitated the development of specific treatment strategies, many of which are in or nearing clinical trials. These strategies include dose intensification, inhibition of P-glycoprotein function, inhibition of cellular glutathione synthesis, and inhibition of cellular DNA repair. The initial results from clinical trials that use these strategies provide reasonable grounds for optimism. In addition, efforts to identify new drugs with activity against resistant cells continue. One such drug, taxol, has significant activity in tumors refractory to conventional therapy. These approaches offer hope that intensive laboratory and clinical efforts ultimately will translate into real improvements in the efficacy of chemotherapy for ovarian cancer.

Alkylating Agents↗

PubMed1993

The biology of ovarian cancer development.

BACKGROUND: In theory, all the cell types that comprise the human ovary have the potential for malignant transformation. The vast majority of malignant ovarian tumors in the human, however, arise from the ovarian surface epithelium. These cells have important functions during reproductive life; they contribute to follicular rupture and by cell division repair the wound that accompanies ovulation. There has been much speculation that the rapid cycles of cell division associated with wound repair contribute significantly to the development of ovarian cancer. Such speculation is based on the observation that ovarian cancer occurs most frequently at the end of a woman's reproductive life and is associated with nulliparity. It is of potential significance that, unlike most epithelia, these cells are not replaced through replenishment stem cells with the development of one end-stage cell and one cell with continued growth potential. Rather, the division of an ovarian surface epithelial cell yields two daughter cells with equal potential for subsequent growth. Thus, all potential mutations as they accumulate are passed on to near-exponentially expanding subsequent generations of cells that can acquire additional mutations that could confer the malignant phenotype. METHODS: We have developed a model to test the hypothesis that repeated cell division by ovarian surface epithelial cells contributes to development of malignancy. In this model, rat ovarian surface epithelial cells are isolated and subjected in vitro to repetitious cell division to mimic in a simple way growth of the surface epithelium in vivo. RESULTS: These cells develop a malignant phenotype based on loss of contact inhibition, the ability for substrate independent growth, tumorigenicity in athymic mice, and cytogenetic changes. CONCLUSIONS: Our data support the involvement of tumor suppressor genes in the development of ovarian cancer.

Animals↗

PubMed1993

Mechanisms of drug resistance in ovarian cancer.

Alkylating agents, natural products and platinum complexes are the primary chemotherapeutic agents used in the treatment of patients with ovarian cancer. Resistance frequently develops to all three classes of drugs and can be functionally separated into distinct biochemical pathways: (1) relative dose intensity plays a role in resistance to platinum complexes and to a lesser degree with alkylating agents; (2) induction of the membrane P-170 glycoprotein confers resistance to natural products and due to the potential usefulness of Taxol (a natural product extracted from the bark of yew trees), this mechanism of resistance may become more clinically relevant in the future; (3) increased levels of cellular glutathione (GSH) and glutathione S-transferases are important in the detoxification of alkylating agents and platinum complexes; and (4) increased DNA repair also is characteristic of resistance to platinum complexes and alkylating agents. Clinical trials have been initiated with agents that may inhibit the biochemical mechanisms of acquired drug resistance. Clinical trials are already in progress with alkylating agents combined with inhibition of GSH biosynthesis (i.e., buthionine sulfoximine) or enzymatic inhibitors of glutathione S-transferase activity (i.e., ethacrynic acid). Furthermore, the combination of aphidicolin, an inhibitor of DNA repair, together with platinum complexes also soon will be clinically tested based on promising results in preclinical models of ovarian cancer. Ovarian cancer is a disease of the elderly. Advances in the pharmacology of platinum compounds and in our understanding of the mechanisms of drug resistance should permit these patients to receive increasingly more effective chemotherapy.

Alkylating Agents↗

PubMed1993

Clinical reversal of drug resistance in ovarian cancer.

Human ovarian cancer cell lines and relevant in vivo model systems have been used to identify mechanisms of resistance associated with alkylating agents and platinum compounds. Drug resistance in ovarian cancer is multifactorial and it is probable that additional mechanisms than those already identified may be responsible for clinical drug resistance. Presently drug transport, increased inactivation in the cytosol, increased repair of damaged DNA, and alterations in signal transduction pathways have been shown to account for resistance of alkylating agents and platinum compounds. Clinical trials have been initiated with agents such as buthionine-sulfoximine, an inhibitor of glutathione biosynthesis which decreases the ability of resistant cells to inactivate platinum compounds prior to their interaction with DNA. In a phase I trial, it has been demonstrated that glutathione levels can be depleted both in normal tissues and tumor biopsies from drug-resistant ovarian cancer patients. Additional clinical trials are needed to determine the effect that glutathione reduction has upon response rate to alkylating agents and platinum compounds. Ultimately, successful modulation of drug resistance may require a combination of agents which inhibit multiple critical biochemical sites. In addition, an understanding of the mechanisms associated with antineoplastic drug resistance may lead to novel therapeutic strategies aimed at preventing the emergence of clinically relevant resistance.

Alkylating Agents↗

PubMed1993

A comparison of clonogenic, microtetrazolium and sulforhodamine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines.

An assay based upon quantitative staining of cellular protein by sulforhodamine B (SRB) has recently been adopted by the NCI for large-scale screening of new drugs. However, there are few data available regarding whether the SRB assay is comparable to other established methods. Cisplatin cytotoxicity was determined in 16 human ovarian carcinoma cell lines by both SRB and clonogenic assays, and by microtetrazolium (MTT) assay in seven cell lines. Cell lines were derived from untreated patients (some of which were selected for cisplatin resistance in vitro) and from patients clinically refractory to cisplatin-based chemotherapy. There was excellent linear correlation between SRB staining and cell number in all cell lines (r = 0.972-0.999). IC50 values obtained by the SRB and clonogenic assay (r = 0.824, P = 0.000022) were highly correlated, although values obtained in the SRB assay were uniformly higher. IC50 values obtained by SRB assay also correlated well with results obtained by MTT assay (r = 0.906, P = 0.0010). Overall, the SRB assay permitted rapid and reliable assessment of cisplatin sensitivity in these cell lines and compared favourably with clonogenic and MTT assays.

Cell Survival↗

PubMed1993

Advanced ovarian cancer. Drug resistance, supportive care and dose intensity.

BACKGROUND: Both intrinsic and acquired drug resistance occur in ovarian cancer. Much work on in vivo or in vitro, obtained drug resistance has been done and this knowledge is presently being converted into clinical studies. MATERIALS AND METHODS: The review focuses on the detoxifying system, MDR (multidrug resistance), and supportive care in relation to dose intensity. RESULTS: In vitro models suggest that the amount of glutathione, glutathione S-transferase activity or metallothioneins could play a role in the outcome of chemotherapy treatment. The results of human tumour samples studies however do not support this idea. Expression of the cell membrane P-glycoprotein in tumour cells appears in vitro to be an important adverse prognostic factor concerning the effect of natural products. Again the results in human tumour samples vary. The supportive agent sodium thiosulphate protects against cisplatin induced nephrotoxicity, but the exact role of sulphur compounds in ameliorating the neurotoxicity is not yet established. The neuropeptide Org 2766 may be a possible neuroprotector. Hemopoietic growth factors such as GM-CSF, G-CSF and interleukin-3, protect the bone marrow to varying degrees. Autologous bone marrow transplantation induces high, but often short lasting response rates in chemotherapy resistant patients. CONCLUSIONS: More clinical studies on intervention of drug resistance and on high dose chemotherapy are needed to define the role of these strategies in ovarian cancer.

Adult↗

PubMed1993

Platinum analogues in preclinical and clinical development.

The impact of cisplatin on chemotherapy for solid tumors has led to the synthesis of many molecules with platinum as their central building block. These so-called platinum analogues have been developed with the obvious goals of improving the antitumor activity of cisplatin and hopefully, at the same time, altering the dose-limiting side effects of the prototype drug. At least 10 such molecules are in clinical development, whereas several others are at various stages of preclinical testing.

Animals↗

PubMed1993

Alkylating agents and immunotoxins exert synergistic cytotoxic activity against ovarian cancer cells. Mechanism of action.

Alkylating agents can be administered in high dosage to patients with ovarian cancer using autologous bone marrow support, but drug-resistant tumor cells can still persist. Immunotoxins provide reagents that might eliminate drug resistant cells. In the present study, concurrent treatment with alkylators and immunotoxins proved superior to treatment with each agent alone. Toxin immunoconjugates prepared from different monoclonal antibodies and recombinant ricin A chain (rRTA) inhibited clonogenic growth of ovarian cancer cell lines in limiting dilution assays. When alkylating agents and toxin conjugates were used in combination, the addition of the immunotoxins to cisplatin, or to cisplatin and thiotepa, produced synergistic cytotoxic activity against the OVCA 432 and OVCAR III cell lines. Studies performed to clarify the mechanism of action showed that cisplatin and thiotepa had no influence on internalization and binding of the 317G5-rRTA immunotoxin. Intracellular uptake of [195m]Pt-cisplatin was not affected by the immunoconjugate and thiotepa. The combination of the 317G5-rRTA and thiotepa, as well as 317G5-rRTA alone, increased [195m]Pt cisplatin-DNA adduct levels. The immunotoxin alone and in combination with the alkylators decreased intracellular glutathione levels and reduced glutathione-S-transferase activity. Repair of DNA damage induced by the combination of alkylators and 317G5-rRTA was significantly reduced when compared to repair after damage with alkylators alone. These findings suggest that immunotoxins affect levels and activity of enzymes required for the prevention and repair of alkylator damage.

Antibodies, Monoclonal↗

PubMed1992

AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas.

Amino Acid Sequence↗