Analysis of karyotype, SCE, and point mutation of RAS oncogene in Indian MDS patients

Mansoor, Ahmed; Bharadwaj, Thashi; Sethuraman, Shriram; Chandy, Mammen; Pushpa, VL; Kamada, Nanao; Murthy, P. Balakrishna

doi:10.1016/0165-4608(93)90052-n

Cited by 14 publications

(5 citation statements)

References 38 publications

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“…For frozen tissue, homogenized powder of tissue was prepared in liquid nitrogen and subjected to DNA extraction using phenol:chloroform method as described by us previously 25. Point mutations of K‐ras (codon 12) oncogene were detected by PCR and allele‐specific oligonucleotide hybridization, as per our previously published protocol 25, 26. Briefly, 1 μg of DNA was used to amplify the entire exon 1 of K‐ras oncogene using 3′ and 5′ primers.…”

Section: Methodsmentioning

confidence: 99%

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Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Ahmed

Sheldon

Fruitwala

et al. 2007

Intl Journal of Cancer

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Oncogenic ras is known to inhibit cell death and growth inhibitory genes and activate prosurvival genes. Proapoptotic gene PAR‐4, has been found to be downregulated by oncogenic ras. Since pancreatic tumors harbor a high incidence of K‐ras point mutations, we hypothesized that oncogenic K‐ras might influence the function and expression of PAR‐4. PAR‐4 expression levels were analyzed in 4 established pancreatic tumor cell lines, 10 normal pancreatic tissues, 44 frozen tumor tissues and 25 paraffin‐embedded pancreatic adenocarcinoma samples by Real Time RT‐PCR, Western blot analysis and immunohistochemistry. K‐ras mutational status was analyzed by allele‐specific oligonucleotide‐hybridization. Expression levels of PAR‐4 were correlated with the K‐ras mutational status and clinical characteristics. Further, modulation of endogenous PAR‐4 was tested by transiently expressing oncogenic ras in a wild‐type K‐ras pancreatic cancer cell line, BxPC‐3. Three cell lines with K‐ras mutations showed low levels of PAR‐4 when compared to a normal pancreatic tissue. Of 44 frozen tumors, 16 showed appreciable upregulation of Par mRNA and 27 showed significant downregulation of PAR‐4 mRNA when compared to normal pancreatic tissue and 1 had levels equivalent to normal pancreatic tissue. Of 25 paraffin‐embedded tumors, 9 showed downregulation of PAR‐4 protein and this downregulation of PAR‐4 correlated significantly with K‐ras mutational status (p < 0.00002). In addition, the presence of PAR‐4 mRNA or protein expression in pancreatic tumors correlated with prolonged survival. Transient overexpression of oncogenic ras in wild‐type K‐ras BxPC‐3 cells significantly downregulated the endogenous PAR‐4 protein levels and conferred accelerated growth. Thus, downregulation or loss of PAR‐4 expression by oncogenic ras may provide a selective survival advantage for pancreatic tumors, through inhibition of proapoptotic pathway mediated by PAR‐4. © 2007 Wiley‐Liss, Inc.

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Section: Methodsmentioning

confidence: 99%

“…The membrane was hybridized with radiolabeled oligonucleotide probe with allele‐specific mutation combinations. The membrane was washed and autoradiographed for analysis of mutations as described by us previously 25, 26…”

Section: Methodsmentioning

confidence: 99%

Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Ahmed

Sheldon

Fruitwala

et al. 2007

Intl Journal of Cancer

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“…c-Ki-ras-2 mutation analysis DNA from UK Pan-1 cells was analyzed for point mutations in c-Ki-ras-2 at codon 12 using allele specific oligonucleotide hybridization described previously (Mansoor et al, 1993). DNA from Hs 578N cells was used as a control for wild-type c-Ki-ras-2.…”

Section: Electrophoretic Mobility Shift Assaysmentioning

confidence: 99%

Rap1 reverses transcriptional repression of TGF‐β type II receptor by a mechanism involving AP‐1 in the human pancreatic cancer cell line, UK Pan‐1

Fralix

Zhao

Venkatasubbarao

et al. 2002

Journal Cellular Physiology

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The TGF-beta signaling pathway has potent anti-mitogenic effects in epithelial cells and loss of negative growth regulation is often associated with increased tumorigenicity. The human pancreatic ductal adenocarcinoma cell line, UK Pan-1, which expresses DPC4, is not highly responsive to TGF-beta due to transcriptional repression of TGF-beta type II receptor (RII). Here, we show that UK Pan-1 cells transfected with a plasmid to overexpress rap1 protein (UK/rap1) causes an increase in RII transcription and restores sensitivity to TGF-beta growth inhibition. The overexpression of rap1 was associated with diminished ras signaling as measured by ras binding domain (RBD)-binding assays. Electrophoretic mobility shift assays (EMSA) analysis revealed increased binding of nuclear proteins to a previously identified positive regulatory element (PRE1) of the RII promoter in rap1 transfected cells. Competition with an oligo containing the AP-1 consensus site was able to inhibit this binding of nuclear proteins to the PRE1 region. Further EMSA analysis using antibodies to various AP-1 components revealed that junB antibodies partially depleted the increase in binding to the PRE1 seen in UK/rap1 cells while antibodies to other AP-1 constituents such as c-jun, c-fos, and ATF-1 had no effect on binding. Consistent with this data, transient transfection of UK Pan-1 cells with junB resulted in greater RII transcription (twofold) as measured by RII-luciferase assay. Mutation of the AP-1 site inhibited junB-mediated or rap1-mediated increases in RII promoter activity. These data suggest that rap1 signaling may mediate an increase in RII transcription via increased binding of nuclear factors including junB to the PRE1 region of the RII promoter.

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“…Diagnosis was RA in three relative frequency of FAB categories is unknown and variable cases, RARS in one, RAEB in six, RAEBT in three, CMML in according to the studies; for instance, CMML has been diagsix, and Non-FAB in the remaining six patients. CMML was nosed in 5 to 10% of cases in some studies, 5,17,18 and in up significantly associated with idiopathic MDS (P = 0.03). The to a third or half of the cases in other reports.…”

mentioning

confidence: 90%

“…(P = 0.01). Including cases in the literature, 9,17,18,19,37,38 eight documented cases (4 M/4 F), aged 0-15 years, with monosomy 7 in half, are available. Two del(5q) and two t(8;21) occurred in 88-173 month old…”

Section: Mds With Genetic Backgroundmentioning

confidence: 99%

Forty-four cases of childhood myelodysplasia with cytogenetics, documented by the Groupe Français de Cytogénétique Hématologique

1997

Leukemia

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Forty-four cases of myelodysplasia in children were studied. cellaneous conditions with or without dysmorphia, malfor-A third of the cases were unclassifiable according to the FAB mations, or mental retardation. 1,3,7,8,10,11 system. Twenty-five cases were 'idiopathic' myelodysplastic We present data on 44 cases of childhood myelodysplasia: syndromes (MDS), four were secondary to chemo-and/or radio-25 idiopathic, four secondary, and 15 genetic cases. therapy, and 15 (1/3) were found associated with a congenital genetic disease (such as familial myelodysplasia, chromosome syndrome, or various dysmorphic features). Polyclonality was retained in some cases, even in the 'idiopathic MDS' group, Patients and methods demonstrating that non-malignant myelodysplasia does exist. Sex ratio was well balanced in this study; mean age at diag-A prospective study was designed by us in conjunction with nosis was 4 years; median survival was 3 years; prognosis was the Société d'Hématologie et d'Immunologie Pédiatrique better in girls and in young children; normal karyotypes were (SHIP). Data were, however, collected independently, with associated with a better outcome, monosomy 7 with intermediate survival, and other chromosomal findings, including tri-different inclusion dates for patients. Data from 10 of our 44 somy 8 and 19, with a worse prognosis; refractory anaemia with

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Analysis of karyotype, SCE, and point mutation of RAS oncogene in Indian MDS patients

Cited by 14 publications

References 38 publications

Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Rap1 reverses transcriptional repression of TGF‐β type II receptor by a mechanism involving AP‐1 in the human pancreatic cancer cell line, UK Pan‐1

Forty-four cases of childhood myelodysplasia with cytogenetics, documented by the Groupe Français de Cytogénétique Hématologique

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