Differential Distribution and Intracellular Targeting of mRNAs Corresponding to the Three Calmodulin Genes in Rat Brain: A Quantitative In Situ Hybridization Study

Palfi, Arpad; Vizi, Sándor; Gulya, Károly

doi:10.1177/002215549904700502

Cited by 29 publications

(16 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Hmax values calculated from the hybridized cRNA values measured in ISH experiments involving the use of radiolabeled riboprobes for CaM I, II, and III at medium concentration exhibited a good correlation with those obtained from saturation experiments. Hmax values expressed in cRNA ISH copy no/mm 2 units displayed a region-specific expression pattern characteristic of the multiple CaM genes in the rodent brain, as previously demonstrated (Solà et al 1996;Palfi et al 1999). …”

Section: Hmax Calculation Methodssupporting

confidence: 80%

See 1 more Smart Citation

Calculation of Maximal Hybridization Capacity (Hmax) for Quantitative In Situ Hybridization: A Case Study for Multiple Calmodulin mRNAs

Vizi

Gulya

2000

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

S U M M A R YIn estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CaM) I, II, and III [ 35 S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [ 35 S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm 2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.

show abstract

Section: Hmax Calculation Methodssupporting

confidence: 80%

“…The specificity of the CaM I, II, and III cRNA probes was verified previously by Northern blotting analysis (Palfi et al 1999). Hybridization with medium concentrations of sense probes resulted in very low labeling (see Figures 6D-6F).…”

Section: Control Experiments For In Situ Hybridizationsupporting

confidence: 74%

Calculation of Maximal Hybridization Capacity (Hmax) for Quantitative In Situ Hybridization: A Case Study for Multiple Calmodulin mRNAs

Vizi

Gulya

2000

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Indeed the promoters and 3 0 and 5 0 UTRs of the three genes are quite different suggesting divergent function. Studies of calmodulin expression in neurons and other systems suggest that indeed the three transcripts serve different functions and may code for different pools of calmodulin within the cell [32][33][34]. Western blot analysis simply looks at total calmodulin levels within the cells without regard for which transcript is predominant, or the sub-cellular localization of the protein.…”

Section: Discussionmentioning

confidence: 99%

Calmodulin modulates Akt activity in human breast cancer cell lines

Coticchia

Revankar

Deb

et al. 2008

Breast Cancer Res Treat

View full text Add to dashboard Cite

Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ER-positive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGF-stimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.

show abstract

“…In addition, local CaM concentrations may also be translationally regulated as CaM mRNA is differentially distributed, a process that likely reflects the conservation of three non-allelic mammalian CaM genes encoding identical proteins but distinct 5Ј and 3Ј non-coding sequences. For example, one pool of CaM mRNA, derived from a specific CaM gene (CALM1) is abundant in the apical dendrites of cerebellar pyramidal cells and may give rise to local reservoirs of CaM; however, mRNAs derived from CALM1 and CALM2 genes are found in neurite outgrowths in nerve growth factor-stimulated PC12 cells, and CALM3-derived transcripts reside within the cell body (45)(46)(47). Base upon these factors, and the number and concentrations of CaM-binding proteins, it is possible that CaM availability is rate-limiting for SK channel surface expression (43).…”

Section: Discussionmentioning

confidence: 99%