Dynamic occupancy modelling of Asian elephants (Elephas maximus) reveals increasing landscape use in Nepal.

Large mammals with general habitat needs can persist throughout mixed used landscapes, however, human-wildlife conflict frequently leads to their restriction to protected areas. Conservation efforts, especially for reducing conflicts with humans, can enhance tolerance of humans towards species like Asian elephants (Elephas maximus) in human-dominated landscapes. Here, we examine how elephant use in the Chure Terai Madhesh Landscape (CTML) covering the entire elephant range of Nepal changed between 2012 and 2020 in relationship to protection status and environmental conditions. We systematically surveyed ~ 42,000 km2 of potential habitat, by dividing the study area into 159 grid cells of 15 × 15 km2 and recorded elephant signs during the cool, dry season in three years (2012, 2018 and 2020). We analyzed the survey data in a single-species, multi-season (dynamic) occupancy modeling framework to test hypotheses regarding the influence of environmental conditions and protected area status on landscape use by elephants over time. The best-supported model included protected area effects on initial use, colonization, and detection probability as well as temporal variation in colonization and detection probability. Initial use and colonization rates were higher in protected areas, however elephants increasingly used cells located both inside and outside the protected areas, and the difference in use between protected areas and outside declined as elephants use became prevalent across most of the landscape. While elephant use was patchily distributed in the first year of surveys consistent with past descriptions of four sub-populations, elephant use consolidated into a western and eastern region in subsequent years with a gap in their distribution occurring between Chitwan and Bardiya National Parks. Our manuscript highlights the increasing landscape use by elephants in both protected areas and areas outside protected areas and suggests that management interventions that focus on reducing conflicts can promote greater use of both protected areas and areas outside of protected areas.

Prospective comparison of PCR-based vs late mRNA-based preemptive antiviral therapy for HCMV infection in patients after allo-SCT.

Human CMV (HCMV)-directed preemptive therapy has helped to improve the outcome following allo-SCT. In this study, we evaluated the safety and efficacy of a late mRNA-based (NucliSens CMV pp67) anti-HCMV treatment strategy. A prospective randomized multicenter pilot trial was performed comparing PCR-based, with late mRNA-based preemptive HCMV-directed antiviral therapy in patients after allo-SCT. In all, 133 patients were randomized in three different centers at the time of transplant, 130 of whom are evaluable. Viral screening was performed weekly. Antiviral therapy was initiated at the second consecutive positive PCR result, or at the first detection of late mRNA. The therapy was stopped if clearance of HCMV DNA or late mRNA was demonstrated after 14 days of antiviral therapy. If HCMV infection persisted, antiviral therapy was continued in a reduced dose. The median duration of antiviral therapy during the first treatment episode was 28 days for PCR-, and 19 days for mRNA-screened patients (P<0.02). However, the overall duration of antiviral therapy, as well as the incidence of HCMV disease and the OS at day 100 after transplantation was comparable between the two study groups. We conclude that late mRNA-based anti-HCMV therapy may show comparable safety and efficacy with PCR-based therapy in patients after allo-SCT.

Comparison of the RNA-amplification based methods RT-PCR and NASBA for the detection of circulating tumour cells.

Increasingly, reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT-PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT-PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT-PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT-PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods.

Evaluation of human cytomegalovirus gene expression in thoracic organ transplant recipients using nucleic acid sequence-based amplification.

BACKGROUND: Human cytomegalovirus (CMV) infection is a major cause of morbidity in transplant patients. Early diagnosis and treatment have been shown to improve outcome. We evaluated the suitability of CMV immediate early, early, and late gene expression detected by nucleic acid sequence-based amplification (NASBA) as markers of CMV infection. METHODS: Blood samples were taken immediately before transplant and every one to two weeks after transplantation for 12 weeks from 50 patients undergoing thoracic organ transplantation. CMV-NASBA was performed and results compared with serology, CMV pp65 antigenaemia (CMV-AG) and the development of clinical CMV infection. Patients received "preemptive" anti-CMV therapy with ganciclovir based on the CMV-AG results. RESULTS: CMV immediate early and early gene expression were detected in 87 and 47%, respectively, of patients without other evidence of CMV infection. CMV late gene expression had a sensitivity of 97% for infection (compared with 83% for CMV-AG P=0.06) and a specificity of 93% (compared with 100% P=NS). Late gene expression occurred at the same time as CMV antigenaemia but 1.1 weeks earlier than the threshold of antigenaemia (CMV-AG>10) used to initiate preemptive therapy. CONCLUSION: NASBA provided a standardized tool for the detection of CMV transcripts with a greater sensitivity than the standard antigenemia test. Detection of immediate early and early gene transcripts was not specific for subsequent infection. CMV late gene expression determined by NASBA was an accurate and early marker of CMV infection. Detection of CMV late gene expression could be used to trigger "preemptive" anti-CMV therapy.

Comparison of immunocytochemistry, reverse transcriptase polymerase chain reaction, and nucleic acid sequence-based amplification for the detection of circulating breast cancer cells.

Detection of tumor cells in blood and bone marrow is increasingly used for the staging of patients with breast cancer and to evaluate the presence of tumor cells in peripheral blood progenitor cell collections to be used after high-dose therapy. We evaluated the sensitivity and specificity of three different methods for detection of tumor cells among non-tumor tissue. An immunocytochemical assay using antibodies directed against epitopes of the cytokeratin-19 (CK19) protein and two RNA-based methods: reverse transcriptase polymerase chain reaction (RT-PCR) and Nucleic Acid Sequence-Based Amplification (NASBA) for the same target gene were tested. With all the three methods, false-positive results were observed when peripheral blood mononuclear cells (PBMC) of healthy volunteers were tested. There was no concordance between the RNA-based assays and the immunocytochemical assay. The false-positive results in the RNA-based assays may be due to 'illegitimate expression' of epithelial genes in normal PBMC. The false-positive results in the immunocytochemical assay resulted from background staining of monocytes and granulocytes. This study demonstrates that CK19 is not a suitable target to detect the presence of breast tumor tells in PBMC. To reliably detect circulating tumor cells with RNA methods, the selection of suitable target genes is required, which are highly expressed in tumors but not at all in normal cells of blood and bone marrow. Genes with such characteristics may be identifiable with novel differential display techniques.

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.

Evaluation of a new method for early detection of active cytomegalovirus infections. A study in kidney transplant recipients.

Early detection of active cytomegalovirus (CMV) infection after organ transplantation is necessary to start effective antiviral treatment. In the present study, blood specimens of kidney transplant recipients (n = 38) were monitored for the expression of CMV immediate early (IE) and late (L) mRNA using nucleic acid sequence-based amplification (NASBA). Results were compared with virus isolation, pp65 antigenemia and serology. In patients developing active CMV infection, pp65 antigen and L mRNA were detected simultaneously. At the same time, positive cell culture results could be reported to the clinic. CMV was detected significantly earlier with IE NASBA than with the other assays. However, the specificity of IE NASBA is lower than that of antigenemia, late NASBA and cell culture. Early detection of IE mRNA is especially useful for patients at high risk of developing symptomatic CMV infection in order that early, adequate antiviral therapy may be started. Late NASBA can be used to monitor further development of CMV infection, comparable to antigenemia.

Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA).

NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.

Diagnostic value of monitoring human cytomegalovirus late pp67 mRNA expression in renal-allograft recipients by nucleic acid sequence-based amplification.

The diagnostic value of monitoring human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) after renal-allograft transplantation was evaluated. RNAs were isolated from 489 whole-blood specimens of 42 patients for the specific amplification of the late pp67 (UL65) mRNA. NASBA results were compared to results from the pp65 antigenemia assay, virus isolation by cell culture, and serology. The sensitivity value for NASBA proved to be higher than that for the antigenemia assay (50 versus 35%) for the detection of HCMV infection, while the sensitivity values of cell culture and NASBA were comparable (54 and 50%, respectively). NASBA detected the onset of HCMV infection simultaneously with cell culture and the antigenemia assay. Both the antigenemia assay and NASBA are very specific (100%) and highly predictive (100%) for the onset of HCMV infection. Antiviral therapy with ganciclovir resulted in negative results for cell culture, the antigenemia assay, and NASBA. In conclusion, monitoring HCMV pp67 mRNA expression by NASBA is a highly specific method for the detection of HCMV infection in renal-allograft recipients and is more sensitive than the antigenemia assay. Furthermore, NASBA can be used to monitor the progression of HCMV infections and the effect of antiviral therapy on viral activity.

Three germline mutations in the TP53 gene.

Three germline mutations in the TP53 tumor-suppressor gene are reported, two of which are not reported previously. A missense mutation at codon 265 of TP53 was found in three patients of a family that complied with the definition of the Li-Fraumeni syndrome. A nonsense mutation in codon 306 was found in a woman who had had a rhabdomyosarcoma at age 4 and a subsequent breast cancer at age 22. She was part of a Li-Fraumeni-like family, but the parental origin of the mutation could not be traced. Finally, while screening for somatic alterations in TP53 in a series of 141 sporadic breast tumors, we detected a constitutional missense mutation in codon 235 in a woman diagnosed with breast cancer at age 26 and a recurrence 4 years later. The recurrence, but not the primary tumor, showed an additional missense mutation at codon 245 as well as loss of the wild-type allele. This suggests that the 245 mutation was particularly important for tumor progression and that there might exist heterogeneity in terms of cancer predisposition potential among the various germline TP53 mutations.

Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study.

PURPOSE: To determine whether the clinical course and the response to chemotherapy of patients with advanced adenocarcinoma of the lung depends on the presence or absence of a ras mutation in the tumor. Mutational activation of K-ras is a strong adverse prognostic factor in stage I or II lung cancer and laboratory studies have suggested that ras mutations lead to resistance against ionizing radiation and chemotherapy. PATIENTS AND METHODS: Patients with advanced adenocarcinoma of the lung with measurable or assessable disease received chemotherapy with mesna, ifosfamide, carboplatin, and etoposide (MICE). Archival biopsies, fresh biopsies, or fine-needle aspirations were tested for the presence of ras gene mutations. Associations of ras mutations with clinical characteristics, response to chemotherapy, and survival were studied. RESULTS: The presence or absence of ras gene mutations could be established in 69 of 83 patients (83%). A total of 261 courses of MICE were administered to 62 informative patients, 16 of whom were shown to have a K-ras mutation-positive tumor. The frequency of mutations (26%) and the type of mutations closely matched the pattern we have previously reported in operable disease. Patients with a ras mutation in their tumor were more likely to have a close relative with lung cancer, but other clinical characteristics, such as pattern of metastases, response, and survival, were similar between the ras mutation-positive and ras mutation-negative groups. CONCLUSION: Patients with advanced lung adenocarcinoma who harbor a ras mutation may have major responses to chemotherapy and have similar progression-free and overall survival as patients with ras mutation-negative tumors. K-ras mutations may represent one of several ways in which early tumors are enabled to metastasize to distant sites.

A method to monitor mRNA levels in human breast tumor cells obtained by fine-needle aspiration.

A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.

Absence of ras gene mutations in early gastric carcinomas.

The aims of this study were to assess the prevalence and type of activating point mutations at codons 12, 13, and 61 of the Ki-, Ha-, and N-ras genes in a series of early gastric carcinomas in white patients and to correlate these ras gene mutations, if any, with the histological type (Lauren classification), the type of growth pattern, and with the Helicobacter pylori status. Haematoxylin and eosin and Giemsa stained sections from 45 formalin fixed, paraffin wax embedded early gastric carcinomas were used to assess the Lauren type, the type of growth pattern, and the antral H pylori status. DNA was extracted according to standard procedures. Mutations at codon 12 of the Ki-ras gene were examined with a polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method and dot blot hybridisation with allele-specific 32P-labelled oligodeoxynucleotide (ASO) probes. All other ras genes were analysed with specific PCR amplification and dot blot hybridisation with ASO probes. Mutations were detected by overnight autoradiography at -70 degrees C. Some 20 intestinal-type and 25 diffuse-type early gastric carcinomas were seen. According to growth pattern, there were 24 small mucosal type early gastric carcinomas, five superficial spreading type early gastric carcinomas, and 16 penetrating type early gastric carcinomas (four penetrating A type, 12 penetrating B type). H pylori was found in the antral mucosa of 28 early gastric carcinomas (62%). Activating ras gene mutations were not found. It was discovered that activating point mutations at codons 12, 13, and 61 of the Ki-, Ha-, and N-ras genes do not play a part in the development of early gastric carcinomas in white subjects, irrespective of Lauren type. Moreover, differences in biological behaviour between early carcinomas with different types of growth pattern are not related to these ras gene mutations. Finally, H pylori positive and H pylori negative gastric carcinomas cannot be discriminated on the basis of ras gene mutational analysis.

Growth arrest associated changes of mRNA levels in breast cancer cells measured by semi-quantitative RT-PCR: potential early indicators of treatment response.

To find early and sensitive indicators of treatment response in breast cancer, we studied the mRNA levels of proliferation-related genes during growth arrest of the human breast cancer cell lines T47D and MCF7. A sensitive reverse transcriptase-PCR (RT-PCR) technique was used in order to monitor gene expression in small samples of cells. Estrogen-depletion and treatment with tamoxifen effectively induced a G1-arrest in both cell lines, accompanied by a decrease of the mRNA levels of histone H4, cyclin A, cyclin D1, and c-myc. Cyclin A expression decreased most strongly: up to 32-fold within 7 days. The expression of c-fos and WAF1 increased during growth arrest. In conclusion, significant changes of the levels of proliferation-related mRNAs, induced by growth arrest, can be measured in small samples of breast carcinoma cells using RT-PCR. Especially the decrease of the cyclin A mRNA level seems a potential early indicator of clinical response to tamoxifen therapy in breast cancer patients.

Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates.

BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.

Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer.

A series of 54 resected primary non-small-cell lung carcinomas was analyzed for p53 gene mutations and for p53 protein accumulation and the findings were correlated with clinical parameters. Mutations in exons 5 through 8 of the p53 gene were identified by a denaturing gradient gel electrophoresis (DGGE) assay and cycle sequencing, whereas p53 protein accumulation was detected in paraffin-embedded tissue by immunostaining using 2 different murine monoclonal antibodies (MAbs) (BP53-12 and DO7). A p53 gene mutation and/or p53 protein accumulation was found in 37 of 54 tumors. Mis-sense mutations were closely associated with positive immunostaining, which was intense in 15 out of 17 cases with a mutation. In 10 tumors, obvious p53 accumulation was detected in the absence of mutations in exons 5 through 8. Conversely, only one of 8 p53 non-sense mutations led to detectable p53 accumulation. The most frequent single base changes were G --> T transversions and C --> T transitions. The presence of a p53 alteration was not related to age, tumor size, stage or histology. However, we found a significant inverse correlation between p53 alterations and the presence of a K-ras mutation. This was reflected in the overall postoperative survival data: patients with p53 alterations in their tumors tended to have a better prognosis than those without a p53 alteration; however, this difference was lost when cases with a K-ras mutation were omitted from the analysis.

N- and KRAS mutations in primary testicular germ cell tumors: incidence and possible biological implications.

Recently, conflicting results have been reported on the incidence of RAS mutations in primary testicular germ cell tumors of adults (TGCTs). In four studies a low incidence of mutations (less than 15%) in a variety of TGCTs or derived cell lines was found, whereas in two other studies a high incidence of N- or KRAS mutations (over 40%) was shown. A total of 62 testicular seminomas (SE) and 34 nonseminomatous TGCTs (NS) were studied thus far. The largest series consisted of 42 TGCTs, studied on paraffin embedded tissue. We present the results of analysis for the presence of N- and KRAS mutations, in codons 12, 13, and 61, in snap frozen samples of 100 primary TGCTs, comprising 40 SE and 60 NS. Using the polymerase chain reaction (PCR) and allele specific oligonucleotide hybridization (ASO), mutations were found in five SE (three in NRAS and two in KRAS, all codon 12), and in one NS (KRAS, codon 12). To exclude underestimation of the incidence of RAS mutations in TGCTs due to the presence of an excess of wild type alleles in the analyzed sample, a PCR technique preferentially amplifying KRAS alleles with a mutation in codon 12 was applied to all SE. This approach, allowing a 250 times more sensitive assay, resulted in the detection of only one additional SE with a mutation. Based on a critical analysis of published data and on our results from the largest series of frozen samples investigated thus far, we conclude that N- or KRAS mutations are rare and apparently not essential for initiation or progression of TGCTs.

Analysis of p53 status in tonsillar carcinomas associated with human papillomavirus.

Tonsillar squamous cell carcinomas (a total of 14) were examined both for the presence of human papillomavirus (HPV) DNA and for p53 alterations. General primer-mediated HPV polymerase chain reaction (GP-PCR) revealed the presence of HPV DNA in 12/14 cases. Subsequent typing by HPV type-specific PCR and sequence or hybridization analysis of GP-PCR products revealed DNA from HPV 16 in seven cases, from HPV 33 in two cases, and from HPV 7, HPV 16/33 and HPV 33/59 each in a single case. p53 immunohistochemistry performed on nine HPV containing tonsillar carcinomas using polyclonal serum CM-1 showed elevated p53 levels in four cases. These included 3/5 HPV 16 containing carcinomas and the HPV 33/59 containing carcinoma. Analysis of p53 mutations using denaturing gradient gel electrophoresis (DGGE) of GC-clamped PCR products of exons 5 to 8 showed p53 gene alterations in 3/13 cases, including 2/11 HPV positive cases and 1/2 HPV negative cases. The alterations included a silent point mutation within exon 8 of an HPV 16 containing carcinoma, a 1 bp deletion within exon 8 of an HPV 33 containing carcinoma, and a missense mutation within exon 7 of one of the HPV negative carcinomas. There was evident discrepancy between p53 immunohistochemistry and gene analysis. Four HPV containing cases showing elevated p53 levels did not reveal the presence of exon 5 to 8 alterations affecting the amino acid code, suggesting the presence of mutations occurring in other exons or non-mutational p53 stabilization. The data indicate that HPV and elevated p53 can coexist in tonsillar carcinomas and that despite the low frequency of p53 mutations the presence of HPV is not exclusively related to the absence of mutated p53.

BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis.

The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL-2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.

Association of H-ras mutations with adenocarcinomas of the parotid gland.

We have examined 17 adenocarcinomas and 2 mixed tumors of the salivary glands for mutational activation of the oncogenes H-ras, K-ras and N-ras. The presence of mutations was determined by in vitro amplification of gene fragments spanning codons 12, 13 and 61 and the use of mutation-specific oligonucleotide hybridization. ras mutations were present in 3 of 13 adenocarcinomas (23%) of the parotid gland. The mutations were confirmed and characterized by means of cycle sequencing of polymerase chain reaction (PCR) fragments. In all 3 cases, the mutation was an A:T to G:C transition at the second position of codon 61 of the H-ras gene. This rather unusual ras mutation could provide a clue to identify one or more carcinogens involved in the pathogenesis of parotid cancer.