No abstract

Heat shock caused significant changes in intracellular pH (pH1) and intracellular free calcium concentration ([Ca2 k) which occurred rapidly after temperature elevation. pH1 fell from a resting level value at 25°C of 7.38 ± 0.02 (mean ± standard error of the mean, n = 15) to 6.91 ± 0.11 (n = 7) at 35°C. The resting level value of [Ca211, in single Drosophila melanogaster larval salivary gland cells was 198 ± 31 nM (n = 4). It increased approximately 10-fold, to 1,870 ± 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(fO-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]j was reduced to 80 ± 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+1J to 353 4-90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, ClP, and Mg2' were 9.6 ± 0.8, 101.9 + 1.7, 36 + 1.5, and 2.4 + 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pH, and[Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pH; which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.Heating Drosophila melanogaster tissues or cultured cells from 25 to 35°C results in the rapid activation of heat shock genes and the selective translation of heat shock mRNA into protein, whereas all other transcription and translation are abruptly halted (1). This response to heat is a nearly universal phenomenon, occurring in procaryotes and in most eucaryotic cells. In addition, the heat shock proteins themselves and the mechanism of their induction have been highly conserved during evolution (1). These features have made the heat shock response one of the most widely used model systems for the study of eucaryotic gene expression.Heat shock genes can be activated by a wide variety of chemical agents, including oxidizing agents, heavy metals, ionophores, and amino acid analogs (1). A central question is whether these agents act in separate ways or whether they act on a common pathway for the expression of heat shock genes. The findings that polytene chromosome puffing at heat shock gene loci can occur within minutes after heating and that synthesis of heat shock mRNA is not affected by protein synthesis inhibitors both suggest that the induction pathway involves preexisting proteins which are activated by some physiological stimulus during a heat shock.Several lines of evidence suggest that the physiological stimulus may be provided by ionic changes in the cytoplasm of heat-shocked cells. Most notable is that treating cells with the ionophores valinomycin, dinactin, A23187, or ionomycin will result in heat shock protein synthesis (1, 25). Changes in plasma membrane fluidity, alterations in permeability to small molecules, and loss of membrane-bound enzyme activities have been shown to occur during heat shoc...

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.

A recently established model for local breast cancer recurrence using the 13762NF rat mammary adenocarcinoma was used to evaluate biologic and biochemical properties related to clinical outcome for this class of tumors. Sublines isolated from local tumor regrowths following surgical resection differed from each other and from the 'parental' cell lines for multiple phenotypes, including metastatic propensity. Local recurrence- and primary tumor-derived sublines were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), lectin binding to electrophoretically separated proteins, and lactoperoxidase-catalyzed cell surface iodination; and differential protein patterns were compared to tumor progression and metastatic potential. 2D-PAGE revealed several quantitatively different spots which correlated with lung colonization potential. In particular, quantities of an apparently unique, non-cell-surface protein, P50.9 (Mr approximately 50,900, pI approximately 7.3) correlated inversely with metastatic propensity, suggesting that it may be associated with, among other possibilities, the negative regulation of the metastatic phenotype. P50.9 was unrelated to four similarly sized metastasis-associated proteins--tumor autocrine motility factor; the rat analog of tumor suppressor, p53; rat cytokeratin 14 or procathepsin D--as determined by amino acid analysis. A major wheat germ agglutinin binding sialoglycoprotein, gp93 (Mr approximately 93,000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 greater than 'primary' tumor-derived lines (sc1, sc3) much greater than local recurrence-derived lines (LR1, LR1a, LR3, LR4, LR5, LR6)]. Besides cell surface glycoprotein losses, two of six local recurrence-derived sublines expressed a wheat germ agglutinin-binding sialoglycoprotein, gp110 (Mr approximately 110,000), previously undetected on any of the other cell lines including the parental populations. gp110 was found in LR3 and LR6 which were relatively highly metastatic; however, correlation with metastatic potential failed because gp110 was not present on the metastatic parental cell line, MTF7. These results demonstrate specific quantitative and qualitative protein differences associated with the selection of locally recurrent mammary tumors.